Quantitative method for functional C1 esterase inhibitor (FC1-INH)

Abstract

Provided herein are methods for quantifying fCl-INH from dried blood spots. Such methods may include spotting and drying a blood sample on a support member, extracting protein from the dried blood sample, and measuring the level of fCl-INH in the extracted protein.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 834,461, filed April 16, 2019, and U.S. Provisional Application No. 62 / 932,011, filed November 7, 2019, under 35 U.S.C. §119(e); the entire contents of each are incorporated herein by reference. Background Art

[0003] Hereditary angioedema (HAE) is a rare autosomal dominant disease caused by a genetic defect or dysfunction of the plasma protein C1 esterase inhibitor (referred to as C1 inhibitor, C1-INH). HAE is characterized by recurrent attacks of angioedema, usually involving the face, skin, intestines and / or airways. These symptoms can occur in other diseases, which complicates the diagnosis of HAE. HAE can be divided into two main types. Type I is characterized by low levels of C1-INH protein, accounting for 85% of the incidence of HAE, while Type II is characterized by low functional C1-INH (fC1-INH) but normal or elevated C1-INH protein levels, accounting for about 15%. In untreated type I and type II patients, typical fC1-INH, when measured in units / ml, is 5-30% of normal levels.

[0004] The assay currently available for measuring fC1-INH is a plasma-based assay. A commonly used test is a chromogenic assay, which uses a synthetic C1-esterase-specific substrate to indirectly measure the inhibition of C1-INH on C1-esterase activity in a test sample. Another commonly used assay is an ELISA assay, which measures the functional binding of C1-INH to complement protein component C1s. However, it is usually inconvenient to carry out plasma-based assays for samples to be transported to and stored in a centralized testing laboratory.

[0005] There is great interest in developing a simple and user-friendly method that can be run in a central laboratory to measure fC1-INH in biological samples. Summary of the invention

[0006] The present disclosure is based, at least in part, on the development of a simple, sensitive, and selective assay involving the use of dried blood spots containing a functional plasma C1-esterase inhibitor (fC1-INH) for analysis by measuring, for example, a substrate product derived from a C1-esterase enzymatic reaction (C1s substrate product) via liquid chromatography-mass spectrometry (LC-MS).

[0007] Therefore, one aspect of the present disclosure provides a method for determining the level of fCl-INH in a dry blood sample, the method comprising: (i) spotting a blood sample from a subject on a support member; (ii) drying the blood sample on a support member to form a dry blood spot; (iii) extracting protein from the dry blood spot of (ii); (iv) measuring the level of fCl-INH in the protein extracted in (iii) (if present). This measurement can be performed for a calibration curve or compared with a calibration curve. In some examples, the calibration curve can be prepared by serially diluting whole blood from a normal subject with a buffer or other appropriate component that does not contain C1-INH.

[0008] In some examples, step (ii) of drying the blood spot can be performed at room temperature for 3 hours. In some examples, the support member is filter paper. Alternatively or additionally, step (iii) of extracting protein from the dried blood spot can be performed by incubating the dried blood spot with bovine serum albumin (BSA) / PBS buffer for at least 3 hours.

[0009] In any of the methods disclosed herein, the step (iv) of measuring the level of fC1-INH may include (a) incubating the extracted protein with complement component 1s (C1s) and a C1s substrate to produce a reaction mixture; (b) measuring the level of the C1s substrate product produced in step (a); (c) determining the level of fC1-INH in the dried blood spot based on the level of the C1s substrate product measured in step (b). In some examples, the C1 substrate product can be measured by liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0010] In some embodiments, step (a) of incubating the extracted protein with complement component 1s (C1s) and a C1s substrate can be performed by incubating the extracted protein with C1s to produce a reaction mixture, and then incubating the reaction mixture with the C1s substrate to produce a C1s substrate product. In some cases, the level of the C1s substrate product can be measured by liquid chromatography-mass spectrometry. Exemplary C1s substrates include, but are not limited to, N α -Benzyloxycarbonyl-Lys-thiobenzyl ester, which produces the C1s product N α -benzyloxycarbonyl-L-lysine (cbz-Lys).

[0011] Any of the methods described herein may also include obtaining a blood sample (e.g., a whole blood sample) from a subject (e.g., a human subject). In some embodiments, the human subject may be a human patient suffering from or suspected of suffering from a C1-INH deficiency-mediated disorder such as hereditary angioedema (HAE), such as type I HAE or type II HAE.

[0012] The methods described herein may also include determining whether the subject suffers from HAE. Compared to the control, a decrease in fCl-INH levels (associated with an increase in C1s substrate product levels) indicates that the subject suffers from a condition mediated by plasma kallikrein (pKal) and / or C1-INH deficiency, such as HAE disclosed herein. Any method described herein may further include (a) identifying appropriate treatment for the subject based on the level of functional C1-INH; (b) identifying the subject as a candidate for treating the disease based on the fCl-INH level determined by the method disclosed herein; or both.

[0013] Any method described herein may also include administering a therapeutic agent to a subject identified as being at risk of a C1-INH defect-mediated disorder or suffering from a C1-INH defect-mediated disorder by any method disclosed herein. In some embodiments, the therapeutic agent may be a plasma kallikrein (pKal) inhibitor, a bradykinin B2 receptor antagonist, or a C1 esterase inhibitor. Examples include, but are not limited to, icaraltide, lanadelumab, icatibant, or a C1 esterase inhibitor derived from human plasma.

[0014] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will be apparent from the following drawings and detailed description of several embodiments, and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram illustrating an exemplary dried blood spot (DBS) based fCl-INH liquid chromatography-tandem mass spectrometry (LC-MS / MS) assay for analysis of the C1s product (cbz-Lys).

[0016] Figure 2 is an exemplary ion chromatogram illustrating the fragmentation pattern of the cbz-Lys parent ion under collision induced dissociation (CID). The most intense peak at m / z 91 was selected as the product ion for detection.

[0017] Figure 3 Included are exemplary ion chromatograms showing cbz-Lys and internal standard from a neat buffer solution as a control (panel (1)); the enzyme reaction from a DBS extract in a surrogate matrix comprising a mixture of red blood cells and a BSA solution in an optimal volume ratio (panel (2)); and the enzyme reaction of a DBS extract from a real whole blood sample from a healthy individual (panel (3)).

[0018] Figure 4 is a calibration curve showing fC1-INH measurement in DBS.

[0019] Figure 5It is used to measure the C1s product (N α Schematic diagram of an exemplary dried blood spot (DBS)-based fCl-INH liquid chromatography-tandem mass spectrometry (LC-MS / MS) assay for cbz-Lysine.

[0020] Figures 6A-6D Exemplary ion chromatograms of representative samples are shown. Fig. 6A Cbz-Lys in pure buffer solution and an internal standard are shown as controls. Figure 6B cbz-Lys and internal standard in blank surrogate matrix are shown. Figure 6C cbz-Lys and internal standard in pooled real blood are shown. Fig.6D cbz-Lys and internal standard in pooled healthy blood spiked with 500 mU / mL C1-INH are shown. mU / mL is milliunits of functional C1-INH level per milliliter.

[0021] Figure 7 is a representative calibration curve showing the measurement of fC1-INH in DBS.

[0022] Figure 8 is a graph showing fC1-INH levels in samples from healthy subjects (n=103) and HAE patients (n=24). mU / mL is milliunits of functional C1-INH level per milliliter.

[0023] Fig. 9 are photographs showing DBS samples (points AD) with punches (N=3 or =6) collected from different locations for analysis.

[0024] Detailed Description

[0025] Genetic defects in the C1 inhibitor protein (C1-INH) cause hereditary angioedema (HAE). HAE patients suffer from acute attacks of painful edema, often caused by unknown triggers.

[0026] C1 esterase (C1s) inhibitor (C1-INH) is a normal component of human plasma and belongs to the group of serine protease inhibitors (serpins), which includes antithrombin III, α1-proteinase inhibitor, α2-antiplasmin, and heparin cofactor II. Like other inhibitors in this group, C1-INH has important inhibitory potential on several major cascade systems in the human body, including the complement system, the intrinsic coagulation (contact) system, the fibrinolytic system, and the coagulation cascade. C1-INH is usually activated during inflammatory processes and inactivates its substrates by covalently binding to reactive sites. C1-INH is the only known inhibitor of complement component 1 (C1r), C1s, coagulation factor XIIa, and kallikrein subcomponents. In addition, it is the main inhibitor of coagulation factor XIa in the intrinsic coagulation cascade.

[0027] HAE can be divided into two main types. Type I is characterized by low levels of C1-INH protein, accounting for 85% of HAE incidence, while Type II is characterized by low functional C1-INH (fC1-INH) but normal or elevated C1-INH protein levels, accounting for about 15%. In untreated type I and type II patients, typical fC1-INH, when measured in units / ml, is 5-30% of normal levels. Accurate and reliable measurement of fC1-INH in patient samples is the basis for HAE diagnosis.

[0028] Currently, two types of plasma tests are used to determine the levels of fC1-INH. The first assay is a chromogenic assay that indirectly measures the inhibition of the activity of the target protease C1s by C1-INH in the test sample using a synthetic C1s-specific substrate. The second test is an ELISA assay based on the functional binding of C1-INH to the complement protein component C1s.

[0029] At present, serum or plasma levels of fC1-INH activity and protein expression are recommended together with complement component 4 (C4) levels for HAE type I / II diagnosis, where fC1-IN activity is the most critical test (see, e.g., Maurer M et al., Allergy (2018) 73: 1575-96). Conventional methods for measuring fC1-INH activity include chromogenic methods or complex formation immunoassays. The chromogenic method combines a synthetic C1s substrate to measure the inhibitory activity of C1-INH protein in a plasma sample, wherein no color formation confirms the inhibition induced by C1-INH. However, the complex formation immunoassay method detects C1-INH-C1s complex formation without directly measuring C1-INH inhibitory activity (see, e.g., Li HH et al., J Allergy Clin Immunol Pract (2015) 3: 200-5).

[0030] C1-INH and C4 antigen levels are usually measured by turbidimetry. These assays require immediate processing and proper storage of blood samples in the physician's office and are not standardized, cost-effective, or readily available in all geographic regions. To date, diagnostic rates range from 5-10% in China, Mexico, Japan, and South Korea to 75-80% in the United States and Western Europe (unpublished data). There is great interest in developing simple and standardized methods for measuring fC1-INH in central laboratories.

[0031] Dried blood spot (DBS)-based assays have been widely used for newborn screening or patient diagnosis of a variety of genetic diseases, such as lysosomal storage diseases (LSDs) (19-23). ​​Results from multiple reports have shown that enzyme activity can be retained in DBS samples (2,24). DBS has unique advantages over conventional plasma or serum assays. First, DBS sampling (e.g., using a finger prick) is less invasive and does not require large amounts of blood or any laboratory equipment. Second, DBS samples can be transported and stored at ambient temperature for long periods of time without significant loss of enzyme activity or genetic information. These advantages enable DBS samples to be prepared in a physician's office and tested in a centralized laboratory.

[0032] The present disclosure describes an assay for measuring functional C1 inhibitor (fC1-INH) levels in dried blood spots (DBS). This DBS-based test has several advantages over plasma-based chromogenic or ELISA assays, such as small blood volume, greater stability of the analyte after drying on filter paper, convenient sample transportation and storage, and / or allows testing of patient samples in centralized laboratories.

[0033] I. Methods for detecting functional plasma esterase C1 inhibitor (fC1-INH) levels

[0034] The present disclosure relates to methods for determining fCl-INH levels by robust liquid chromatography-tandem mass spectrometry (LC-MS / MS) using dried blood spots (DBS) prepared from whole blood samples of patients. Such methods can be used to diagnose conditions mediated by C1-INH deficiency, such as HAE. The method can involve preparing a DBS sample, extracting protein from the DBS sample, incubating the protein extract with complement component 1s (C1s), and reacting with a C1s substrate to produce a C1s substrate product, which can be measured, for example, by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0035] (i) Sample preparation

[0036] Any sample that may or may not contain C1-INH (e.g., fC1-INH, non-functional C1-INH, or both) can be analyzed by the methods described herein. As used herein, "sample" refers to a composition that may include an analyte of interest (fC1-INH in this case). The sample may include tissue, blood, plasma, or serum from a subject. The term "sample" may include an initial untreated sample taken from a subject and a subsequently treated sample, such as a partially purified or preserved form, for example, by immunoprecipitation. Exemplary samples include blood, plasma, serum, tears, or mucus. In other examples, the sample may be a composition from an in vitro assay.

[0037] In some embodiments, the sample is a body fluid sample, such as a serum or plasma sample. In some embodiments, the sample is a biological sample obtained from a subject to be analyzed. "Patient," "subject" or "host" (these terms are used interchangeably) may refer to a human or non-human animal. In some cases, the subject is a human patient who may suffer from, be suspected of suffering from, or be at risk of suffering from a disease associated with a contact system. For example, a human patient may have previously developed HAE or may be at risk of HAE. Such a human patient may have been previously treated or may be in the process of treatment with a drug that targets a component of the contact system (e.g., C1s, plasma kallikrein).

[0038] The biological sample can be a body fluid sample, such as a blood sample. In some examples, the blood sample is a whole blood sample. Whole blood includes red blood cells, white blood cells, platelets and plasma. In some embodiments, the blood sample can be collected from blood vessels (e.g., capillaries, veins and arteries). The whole blood sample used in the methods described herein can be collected and processed using any method known in the art. In some embodiments, the sample is obtained by finger blood sampling (finger prick). In some embodiments, a vacuum blood collection tube can be used to collect the sample.

[0039] The sample of the present disclosure can have any volume sufficient to carry out at least one fCl-INH measurement assay as described herein. In some embodiments, the sample obtained from the subject is between 25 μL-10mL. In some embodiments, the sample or its aliquot for the method described herein is between 100 μL-2mL. In some embodiments, the sample is between 50 μL-100 μL. In some embodiments, the sample is between 50 μL-1mL. In some embodiments, the sample is 50 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.5 mL, 5.0 mL, 5.5 mL, 6.0 mL, 6.5 mL, 7.0 mL, 7.5 mL, 8.5 mL, 9.0 mL, 9.5 mL, or 10.0 mL. In some embodiments, the sample is 60 μL.

[0040] Any sample disclosed herein, such as a whole blood sample, can be collected, and an aliquot of the sample can be removed and spotted on a support member. In some embodiments, the support member containing the sample or an aliquot of the sample is maintained under appropriate conditions (e.g., room temperature) for an appropriate period of time (e.g., at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or longer) to allow a dried spot of the sample to form on the support member.

[0041] The support member can be a membrane, a film, a filter paper, or a dried blood spot card. In some embodiments, the support member can include one or more sample collection areas and, optionally, a wrap-around cover. The support member can fit into a sealable container, such as Bags to facilitate sample transfer. In some embodiments, the support member can be foldable. Any support member described herein may further include one or more areas for recording additional information of the sample (e.g., demographic information of the subject).

[0042] The sample (or its aliquot) can be allowed to dry on the support member under appropriate conditions and within an appropriate time period. In some embodiments, the sample is allowed to dry for about 1-6 hours, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In some embodiments, the sample is dried for about 1-2 hours, 1-3 hours, 1-4 hours, 1-5 hours, 2-3 hours, 2-4 hours, 2-5 hours, 2-6 hours, 3-4 hours, 3-5 hours, 3-6 hours, 4-5 hours, 4-6 hours, or 5-6 hours.

[0043] In some embodiments, the sample is allowed to dry at an appropriate temperature, such as a temperature between 10°C-40°C or a higher temperature. In some embodiments, the sample is allowed to dry at 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or a higher temperature. It is known to those skilled in the art that the drying process can be carried out at a relatively high temperature for a shorter time, and optionally, at a relatively low temperature for a longer time. In some examples, the sample can be allowed to dry at room temperature (about 25°C) for at least 3 hours.

[0044] (ii) Protein extraction

[0045] A dried spot sample prepared as described herein, such as a dried blood sample, can be processed to extract biological materials contained in the sample, such as proteins. In some embodiments, a dried spot sample, such as a DBS sample, can be processed for protein extraction. As used herein, protein extraction refers to extracting proteins or certain portions of proteins contained in a dried sample into an appropriate buffer (e.g., the target protein is soluble in the buffer). In some cases, a dried spot sample (e.g., a DBS sample) present on a sample collection area of ​​a support member can be separated from the rest of the support member by conventional methods (e.g., using a puncher). The separated biological material sample, such as a protein sample, can be transferred to an appropriate container for further protein extraction. Non-limiting examples of suitable containers can be tubes, trays, vials, and multi-well plates. In some examples, a suitable container can be a 96-well plate. One or more dried spot samples, such as DBS samples, can be processed simultaneously.

[0046] Any suitable buffer can be used in the methods described herein. Preferably, the buffer is compatible with the biomaterial to be extracted (e.g., capable of dissolving proteins and preferably retaining protein function). Non-limiting examples of suitable buffers include phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS) or Hanks balanced salt solution (HBSS). In some examples, the buffer can be PBS. In some embodiments, the buffer can include one or more components to promote stability and / or prevent degradation of the biomaterial to be extracted.

[0047] In some embodiments, the buffer used for the extraction step may contain one or more reagents that promote protein extraction (e.g., bovine serum albumin) at an appropriate concentration (e.g., 0.5%). In some embodiments, a support member or a portion thereof containing a dry spot sample (e.g., a DBS sample) may be incubated in a buffer under appropriate conditions that allow complete extraction of the biological material of interest (e.g., fCl-INH) in the sample. In some embodiments, the support member containing the dry spot sample may be incubated in a buffer at an appropriate temperature, such as 25-37° C. (e.g., 37° C.), for an appropriate time, such as 2-5 hours (e.g., 3 hours), to allow sufficient extraction of the biological material of interest (e.g., proteins including fCl-INH), so that the level of fCl-INH can be measured.

[0048] In some embodiments, the buffer / sample mixture can be centrifuged at an appropriate speed, such as 800-2000 rpm (e.g., 1250 rpm) to facilitate extraction of the biological material into solution. The selection of specific extraction conditions, including temperature, extraction time period, use of buffer, centrifugation or no centrifugation, centrifugation speed, etc., can depend on, for example, the biological material to be extracted and the type of support member used to form the dry spot sample. Such information is within the knowledge of those skilled in the art.

[0049] (iii) Measurement of fC1-INH

[0050] A solution containing fCl-INH prepared as disclosed above can be analyzed to determine the level of fCl-INH in the solution. In some cases, the level of fCl-INH is measured by determining the activity of fCl-INH, for example, by a conventional route or method disclosed herein.

[0051] The level of fCl-INH can be measured using appropriate methods known in the art or as described herein. In some embodiments, the level of fCl-INH is measured by a chromogenic assay using a chromogenic substrate that can be cleaved by C1s. In some embodiments, an immunoassay is used to assess the level of a C1s product of interest as described herein. Examples of immunoassays include, but are not limited to, Western blots, enzyme-linked immunosorbent assays (ELISAs) (e.g., sandwich ELISAs), radioimmunoassays, electrochemiluminescence-based detection assays, and related techniques. Assays, such as Western blot assays, may further involve the use of a quantitative imaging system, such as the commercially available LICOR imaging technology (see, e.g., from LI-COR Biosciences). CLx infrared imaging system). In some embodiments, an electrochemiluminescence detection assay or an assay relying on a combination of electrochemiluminescence and patterned array technology (e.g., ECL or MULTI-ARRAY technology assays from Meso Scale Discovery (MSD)) is used.

[0052] As used herein, the term "measuring" or alternatively "detecting" refers to assessing the presence, absence, quantity or amount (which may be an effective amount) of a substance in a sample, including deducing a qualitative or quantitative concentration level of such a substance, or otherwise assessing a value or classification of a subject.

[0053] In some embodiments, a sample containing fCl-INH extracted from any dry spot sample disclosed herein (e.g., a DBS sample) can be incubated with a C1s protease and a C1s substrate. The C1s protease can act on the C1s substrate to convert the C1s substrate into a C1s substrate product, which can then be analyzed to determine the presence and / or level of the C1s substrate product. Alternatively, the C1s substrate can be analyzed to determine the presence and / or level of the C1s substrate remaining after incubation with the C1s protease and a sample containing fCl-INH. Once the C1s protease is inhibited by fCl-INH, the production of the C1s substrate product is reduced or eliminated. As used herein, "C1s substrate product" refers to a cleavage product produced by the protease reaction of C1s on the C1s substrate. In some embodiments, the amount of the C1s substrate and / or the C1s substrate product can be used as an indirect measure of the activity of the C1s protease and / or fCl-INH.

[0054] In some embodiments, a sample containing fCl-INH extracted from any dry spot sample disclosed herein (e.g., a DBS sample) can first be incubated with a C1s protease for an appropriate time under appropriate conditions. The C1s substrate can then be added to the mixture, which can be further incubated for an appropriate period of time under appropriate conditions to allow the production of a C1s substrate product. In some embodiments, the incubation can be performed in the dark. In some examples, the method can include a step of quenching the C1s protease reaction, which can increase the accuracy of the analysis. In some embodiments, the C1s protease reaction can be quenched by diluting (e.g., 1:5-1:20) the C1s reaction mixture in a quenching solution. As used herein, a "quenching solution" refers to a solution used to stop an enzymatic reaction as described herein (e.g., a C1s protease reaction). In some embodiments, the quenching solution may contain a reagent capable of denaturing or inactivating the enzyme. Non-limiting denaturants include alcohols, detergents, strong acids, strong bases, chelating agents, and the like. In some embodiments, the quenching solution can include a detergent (eg, SDS) and / or an alcohol (eg, methanol).

[0055] The C1s substrate used in the methods disclosed herein can be any substrate that can be cleaved by a C1s protease to produce a detectable C1s substrate product. Non-limiting examples of C1s substrates include N α -Benzyloxycarbonyl-Lys-thiobenzyl ester, which after cleavage by C1s protease generates N α -benzyloxycarbonyl-L-lysine (cbz-Lys); and C4, producing C4a and C4b.

[0056] In some embodiments, after the enzymatic reaction is quenched, the sample can be further diluted for subsequent liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. In some embodiments, the sample can be diluted 200 times in an appropriate solution (e.g., 80% MeOH in water (v / v)).

[0057] The resulting C1s substrate product can be analyzed by conventional methods to determine its presence and / or level. In some embodiments, the C1s substrate product can be analyzed by liquid chromatography-mass spectrometry (LC-MS), which is an analytical technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). LC-MS / MS assays can be performed using conventional methods.

[0058] In some cases, the mass spectrometer used to analyze the C1s substrate product can be operated in a selective reaction monitoring (SRM) mode. The SRM mode is a highly sensitive and selective method for tandem mass spectrometry, in which ions of a specific mass are selected in the first stage of the tandem mass spectrometer, and the ion products of the fragmentation reaction of the precursor ion are selected for detection in the second mass spectrometer stage. The SRM mode can be used for targeted quantification by a mass spectrometer. After ionization, for example, using an electrospray source, the precursor is first separated to obtain a large number of ion groups that are mainly expected substances (e.g., parent ions). The group is then fragmented to produce product ions, and its signal abundance indicates the abundance of the product of interest in the sample. In some embodiments, the cbz-Lys parent ion can be at m / z281 and the cbz-Lys product ion selected for detection can be at m / z 91. As described in the example, a suitable instrument, such as an ABSciex 5500 or 6500QTrap mass spectrometer, can be used and the differences between different samples can be evaluated after method optimization. As described herein, individual whole blood samples can be prepared for LC-MS / MS analysis.

[0059] The level (e.g., concentration) of the C1s substrate product (e.g., represented by AUC), and thus the fC1-INH concentration, can be determined by conventional methods. A calibration or standard curve (e.g., a four-parameter logistic calibration curve) can be generated by plotting the concentration of fC1-INH versus the peak area ratio of the analyte (e.g., cbz-Lys) and the internal standard (e.g., the concentration of Nε-benzyloxycarbonyl-L-lysine-2,6,6-d3). The calibration curve can be fitted to an equation, for example:

[0060]

[0061] Where y refers to the peak area ratio; x refers to the sample concentration; and A, B, C, and D are curve fitting parameters.

[0062] In some embodiments, the accuracy and reproducibility of the LC-MS / MS method can be verified by analyzing calibration standards prepared using the methods described herein. In some embodiments, the level (e.g., concentration) of fCl-INH in a sample can be calculated by an equation (e.g., Equation 1). These results indicate that the level of fCl-INH prepared by the methods described herein, represented and calculated by the level of C1s substrate product in a sample, is a reliable biomarker for HAE diagnosis.

[0063] In some cases, a calibration curve can be constructed using the peak area ratio of the analyte of interest (C1s characteristic product) to the internal standard. As used herein, "internal standard" refers to a chemical substance added in a constant amount to a test sample, a quality control (QC) sample, a blank, and a calibration standard to correct for the loss of the analyte during sample preparation, thereby improving the accuracy of the analyte analysis. In some embodiments, the internal standard is Nε-benzyloxycarbonyl-L-lysine-2,6,6-d3 or another stable isotope labeled C1s substrate product. In some embodiments, the calibration curve can be used for quantitative analysis.

[0064] In addition, a series of calibration standards can also be prepared, and LC-MS / MS analysis is performed with sample, using sample as the calibration product of determination and for quality control.As used herein, calibration standards refer to whole blood or solution containing the analyte of concern or material (e.g., fCl-INH in the present disclosure) of known amount.Calibration standards can be used to test the material of known concentration in the same manner as sample, to ensure that the test system accurately measures the sample in the entire reportable range.Calibration standards can be prepared by adding the material of concern (e.g., fCl-INH) of various known amounts to a solution that does not initially contain or depletes the material of concern.In some examples, the material of concern (e.g., fCl-INH) can be added to a biological sample similar to a test sample (e.g., a whole blood sample) to simulate a test sample (e.g., a surrogate matrix).In one example, calibration standards can be prepared by mixing fCl-INH (e.g., purified fCl-INH) with the substitute blood exhausted by fCl-INH. As used herein, fC1-INH depleted replacement blood can be prepared by: (i) obtaining and / or converging fresh whole blood from an appropriate population by a suitable method (e.g., evacuating a blood collection tube), the population is ideally the same species as the subject (e.g., healthy individuals, males and / or females); (ii) removing the supernatant plasma using a suitable method (e.g., centrifugation at 800 rpm for 10 minutes at room temperature) to deplete the endogenous substance of interest in the blood sample (e.g., fCl-INH); (iii) mixing an appropriate volume (e.g., equal volume) of the remaining red blood cells of the confluence with an appropriate buffer (e.g., 4.3% BSA in PBS). The calibration standards and quality control (QC) of the material of interest (e.g., fC1-INH) can be prepared by incorporating various known concentrations of a solution containing the material of interest (e.g., fC1-INH) into the replacement blood depleted of fC1-INH as described herein. As described herein, the calibration standards can be subjected to a method for producing a C1s substrate product prior to LC-MS / MS analysis.

[0065] In a specific example, the method described herein may include: (1) preparing a DBS card from a whole blood sample; (2) extracting C1-INH protein from the DBS card; (3) incubating the extracted protein with complement component C1s; (4) incubating with C1s substrate N α -Benzyloxycarbonyl-Lys-thiobenzyl ester undergoes an enzyme reaction to generate the enzyme product N α -benzyloxycarbonyl-L-lysine (cbz-Lys); (5) followed by N ε -Benzyloxycarbonyl-L-lysine-2,6,6-d3 was used as an internal standard for quantification and cbz-Lys was measured by LC-MS assay.

[0066] II. Application of the dry spot assay

[0067] The assays described herein for measuring fC1-INH in a sample can be used for clinical or non-clinical purposes.

[0068] (i) Disease diagnosis and prognosis

[0069] Diagnosis of a C1-INH deficiency-mediated disorder, such as hereditary angioedema (HAE), can be based on fC1-INH levels in a sample obtained from a candidate subject.

[0070] The assay methods and kits described herein can be used to assess a disease, such as the diagnosis or prognosis of a disease. The assessment may include identifying a subject as being at risk of or suffering from a disease described herein, such as a C1-INH defect-mediated condition, such as HAE (e.g., type I HAE or type II HAE). The assessment may also include monitoring the treatment of the disease, such as assessing the effectiveness of treatment for a C1-INH defect-mediated disease (e.g., HAE). In addition, the assessment may include identifying a disease that can be treated by a pKal inhibitor, such as a C1s inhibitor (e.g., a C1s inhibitor derived from human plasma) or a plasma kallikrein inhibitor.

[0071] A. diagnosis

[0072] In some embodiments, the assays, methods, and kits described herein are used to determine the level of fCl-INH in a biological sample (e.g., a whole blood sample) collected from a candidate subject (e.g., a human patient suspected of having a C1-INH deficiency-mediated disorder (e.g., HAE)). The level of fCl-INH can be determined based on the level of the C1s substrate product produced by the methods described herein (e.g., by a 4-parameter logistic calibration curve). This fC1-INH level can be compared with a predetermined reference value or reference ratio to determine whether the subject suffers from a C1-INH deficiency-mediated disorder, such as HAE, or is at risk of suffering from a C1-INH deficiency-mediated disorder, such as HAE. For example, if the fC1-INH in the candidate subject's sample is or is below a reference value, the subject can be identified as suffering from a C1-INH deficiency-mediated disease, such as HAE, or is at risk of a C1-INH deficiency-mediated disorder, such as HAE.

[0073] Reference sample can be the control level of fCl-INH as described herein.In some embodiments, control level represents the amount of fCl-INH in the control sample (such as whole blood sample) obtained from healthy subjects or healthy subject colony, and the healthy subjects or healthy subject colony preferably have the same species and age as candidate subject.As used herein, healthy subjects are subjects who obviously do not have target disease (for example, the illness mediated by C1-INH defect, such as HAE) or do not have the disease history when measuring fCl-INH level.

[0074] Alternatively, the reference value may be a predetermined value.Such a predetermined characteristic fC1-INH may represent the value of fC1-INH as described herein in a population of subjects not suffering from or not at risk of the target disease.

[0075] The predetermined value can take many forms. For example, it can be a single cutoff value, such as a median or mean value. In some embodiments, a predetermined level can be established based on a comparison group, such as one of the defined groups is known to have a target disease and another defined group is known to have no target disease. Alternatively, the predetermined level can be a range, such as the fC1-INH range in a control population within a predetermined percentile.

[0076] Control value as described herein can be determined by routine techniques.In some examples, control value can be obtained by carrying out conventional method (for example, for obtaining the same determination of fCl-INH level as described herein) to the control sample also described herein.In other examples, the level of fCl-INH can be obtained from the member of the control population and can be obtained by, for example, calculation program analysis result to represent the control level (predetermined level) of fCl-INH level in the control population.

[0077] By comparing fCl-INH concentration as described herein with reference ratio as described herein in the sample obtained from candidate subject, it can be determined whether candidate subject suffers from a disease (for example, HAE) mediated by C1-INH defect or is in the risk (for example, HAE) of a disease mediated by C1-INH defect. For example, if the fCl-INH value in the sample of candidate subject deviates from reference value or ratio (for example, reduced compared with reference value), then candidate subject can be identified as suffering from this disease or is in the risk of this disease. When reference value represents the value range of fCl-INH as described herein in the subject population with target disease, the fCl-INH value falling into the candidate sample of this scope shows that candidate subject has target disease or is in the risk of target disease.

[0078] As used herein, "a reduction value below a reference value" refers to a level of fC1-INH that is below a reference value, such as a predetermined threshold value of fC1-INH in a control sample. The control level is described in detail herein. The reduction value of fC1-INH includes, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more values ​​lower than the reference value of the control sample.

[0079] In some embodiments, the candidate subject is a human patient with one or more symptoms of a C1-INH deficiency-mediated condition, such as HAE. For example, the subject may have edema, swelling, wherein the swelling is entirely or primarily peripheral; urticaria; redness, pain, and swelling in the absence of signs of infection; non-histamine-mediated edema, recurrent swelling episodes, or a combination thereof. In other embodiments, the subject has no symptoms of a C1-INH deficiency-mediated condition at the time of sample collection, no history of symptoms of a C1-INH deficiency-mediated condition, or no history of a C1-INH-mediated condition such as a history of a defect-mediated disease of HAE. In other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0080] B. Identification of appropriate treatment

[0081] In some embodiments, the assay methods and kits described herein can also be used to identify the appropriate treatment of a subject suffering from a C1-INH defect-mediated illness (e.g., type I HAE or type II HAE) or in the risk of suffering from a C1-INH defect-mediated illness (e.g., type I HAE or type II HAE). For example, the fC1-INH level of the subject can be measured using any method described herein and compared with a predetermined value of fC1-INH. If the fC1-INH value of the subject is equal to or lower than a predetermined value, the subject can be treated by a treatment based on a predetermined value of fC1-INH (e.g., a recombinant C1-INH therapeutic agent or other therapeutic agents described herein). In some examples, the subject can be a candidate for a disease treatment based on the level of fC1-INH.

[0082] In some embodiments, the methods described herein further include administering a therapeutic agent to a subject suffering from a C1-INH deficiency-mediated disorder (e.g., HAE) or at risk of suffering from a C1-INH deficiency-mediated disorder (e.g., HAE). Non-limiting examples of therapeutic agents include plasma kallikrein (pKal) inhibitors (e.g., ecallantide, lanadelumab), bradykinin B2 receptor antagonists (e.g., icatibant), and C1s inhibitors (e.g., human plasma-derived C1s inhibitors).

[0083] (ii) Non-clinical applications

[0084] Further, the assay methods described herein may have non-clinical applications, e.g., for research purposes and / or preclinical drug development purposes. Although many diseases associated with C1-INH deficiency have been identified, it is also possible that other diseases are mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein may be used to identify diseases associated with C1-INH deficiency. In some embodiments, the methods described herein may be used to study the mechanism (e.g., discover new biological pathways or processes related to disease development) or progression of a disease.

[0085] In some embodiments, the fCl-INH level determined by the assay method as described herein can be used to develop a novel therapy for the disease associated with the lack of C1-INH. For example, the fCl-INH level as described herein can be measured in a sample obtained from a subject having applied a novel therapy (e.g., gene therapy) or in a sample obtained from an in vitro assay. In some embodiments, the fCl-INH level can indicate the activity of a novel therapeutic agent (therapeutic) in an in vitro test or the efficacy of a novel therapeutic agent in a clinical trial setting.

[0086] III. Kits for performing dry spot assays

[0087] The present disclosure also provides a kit for measuring fCl-INH levels as described herein. Such a kit may include materials for collecting and preparing samples, extracting proteins from samples, components for measuring fCl-INH in samples and / or instructions for determining fCl-INH levels in samples.

[0088] In some embodiments, the kit includes a support member, such as a membrane, filter paper, or dried blood spot card. The selection of an appropriate support member for the method will depend on various factors, such as the number of samples to be evaluated and the method of extracting protein from the sample.

[0089] In some embodiments, the supporting member is a multi-well plate, such as an ELISA plate. In some embodiments, the immunoassay described herein can be performed on a high-throughput platform. In some embodiments, a multi-well plate, such as a plate of 24-, 48-, 96-, 384- or more holes, can be used for high-throughput immunoassays. A separate immunoassay can be performed in parallel in each hole. Therefore, it is generally necessary to use a plate reader to measure multiple holes in parallel to increase the measurement throughput. In some embodiments, a plate reader that can perform parallel imaging of multi-wells (e.g., 4, 16, 24, 48, 96, 384 or more holes) can be used for the platform.

[0090] The kit may also include one or more buffers as described herein, but are not limited to quenching buffer, denaturing buffer, and protein extraction buffer. Examples of buffers include, but are not limited to, PBS, DPBS, HBSS, HEPES, Tris, Tris-HCl, sodium phosphate, potassium phosphate, and potassium chloride.

[0091] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of how to use the components included in the kit to measure fCl-INH levels in a biological sample collected from a subject (e.g., a human patient).

[0092] Instructions for use associated with the kit typically include information about the amount of each component and the appropriate conditions for performing the methods described herein. The components in the kit may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0093] The label or package insert indicates that the kit is used to assess the level of fC1-INH in one or more samples. Instructions for implementing any of the methods described herein may be provided. In some embodiments, the kit may include a sealable container (e.g., bag).

[0094] The kit of the present disclosure is packaged in an appropriate manner. Appropriate packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.

[0095] The kit may optionally provide additional components, such as explanatory information, such as controls and / or standard or reference samples, such as to generate a calibration curve. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article comprising the contents of the above-mentioned kit.

[0096] Without further elaboration, it is believed that those skilled in the art can make the most of the present invention based on the above description. Therefore, the following specific embodiments should be interpreted as being merely illustrative and not limiting the remainder of the present disclosure in any way. For the purposes or themes mentioned herein, all publications cited herein are incorporated by reference. Example

[0097] Example 1 ： DBS-based LC-MS / MS fC1-INH assay

[0098] This example describes a dried blood spot (DBS) based LC-MS / MS analysis for measuring functional C1-inhibitor (fC1-INH) in blood samples. Figure 1 An exemplary protocol for this assay is illustrated.

[0099] The method is designed to determine fC1-INH levels in human whole blood samples. The method involves preparation of calibration standards, quality control (QC) and DBS samples, protein extraction from DBS samples, incubation of protein extracts with complement component 1s (C1s), incubation with the C1s substrate N α -Benzyloxycarbonyl-Lys-thiobenzyl ester to produce N α -Benzyloxycarbonyl-L-lysine (cbz-Lys), and then measured cbz-Lys using liquid chromatography-tandem mass spectrometry (LC-MS / MS). An AB Sciex QTrap 6500 mass spectrometer was operated under optimized conditions in selected reaction monitoring (SRM) mode to detect cbz-Lys and an internal standard (Nε-benzyloxycarbonyl-L-lysine-2,6,6-d3) in positive ion mode.

[0100] Experimental procedures :

[0101] (i) Preparation of calibration standards and quality controls (QCs)

[0102] Calibrators and quality controls (QC) were prepared from replacement blood depleted of C1-INH protein. In brief, fresh whole blood from healthy individuals (male and female) was pooled and centrifuged at 1200 × g and room temperature for 10 minutes. The supernatant plasma was discarded and the resulting red blood cells were washed once with phosphate buffered saline (PBS) solution. Replacement blood without C1-INH was prepared by mixing equal volumes of pooled red blood cells and 4.3% bovine serum albumin (BSA) in PBS. In order to prepare calibration standards and quality controls (QC), C1-INH solutions of different concentrations were added to the replacement blood.

[0103] (ii) Preparation of DBS samples

[0104] After collecting the whole blood sample, a maximum of 60 μL aliquots were deposited on the filter paper spots of DBS cards (903 protein preservation cards, Whatman). The DBS cards were bent so that the back was not in contact with any surface to avoid blood soaking through the filter paper and causing losses. The cards were dried for at least 3 hours (h) and stored at room temperature.

[0105] To prepare calibrators, QCs, and test samples, a DBS (GE Health Care Life Science, Whatman) hole punch was used to punch 3.0 mm holes, and the DBS samples were transferred to 500 μL 96-well plates (Eppendorf Protein Lobind). The DBS samples were extracted by incubating with 100 μL of 0.5% BSA in PBS buffer at 37°C for 3 hours in an incubator running at 1250 rpm. An aliquot (20 μL) of the extracted sample was transferred to another 96-well plate and mixed with 50 μL of a freshly prepared 0.5 μg / mL C1s solution in 0.5% BSA in PBS. After incubation at 800 rpm and 37°C for 1.5 hours, 105 μL of substrate solution (N α -Benzyloxycarbonyl-Lys-thiobenzyl ester) and an internal standard (Nε-benzyloxycarbonyl-L-lysine-2,6,6-d3) were added, and the enzyme reaction was allowed to proceed by incubation at room temperature in the dark for 40 minutes. The reaction was quenched by transferring 50 μL of the reaction solution to 450 μL of 0.1% SDS in MeOH / water (80 / 20, V / V). The above sample was further diluted 200 times with MeOH / water (80 / 20, V / V) and then subjected to LC-MS / MS analysis.

[0106] (iii)LC-MS / MS analysis

[0107] Separation of unreacted substrate, substrate products (analyte, cbz-Lys) and internal standard was achieved on a Waters AcquityUPLC using a reverse phase column (Waters Xbridge Protein BEH C4, 3.5 μm, 2.1×50 mm) set at 30° C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The flow rate was 0.3 ml / min, and the mobile phase gradient included gradient steps between the following time points (time, % B): 0 min, 4%; 0.5 min, 4%; 3.5 min, 12%; 3.6 min, 70%; 4.6 min, 70%; 4.7 min, 4%; 5.5 min, 4%. The autosampler temperature was set to 4° C. and 3 μL of sample was injected in partial-loop LC injection mode. Analyte and internal standard signals were acquired using positive ions formed by electrospray ionization on an AB Sciex QTrap 6500 mass spectrometer operating in selected reaction monitoring (SRM) mode with optimized settings for the detection of cbz-Lys and the internal standard (Nε-benzyloxycarbonyl-L-lysine-2,6,6-d3).

[0108] The optimized MS parameter settings were as follows: curtain gas, 20; collision gas, medium; ion spray voltage, 4000; temperature, 550; ion source gas 1, 50; ion source gas 2, 50; declustering voltage, 120; entrance voltage, 10.0; collision energy, 26.0; collision cell exit voltage, 12.0. The analytes and internal standards were monitored using SRM ions from m / z 281.2 to m / z 91.0 and m / z 284.2 to m / z 91.0, respectively.

[0109] (iv) Data analysis

[0110] The peak areas of the analytes and internal standards were determined by Analyst software. A 4-parameter logistic calibration curve was constructed using SoftMax Pro 7.0 with the peak area ratio of the analytes to the internal standard and the concentration of C1-INH spiked into the standards. The fC1-INH level in the sample was calculated based on the following equation.

[0111]

[0112] Where y is the peak area ratio; x is the sample concentration; A, B, C, and D are curve fitting parameters. In addition, Excel was used to calculate the accuracy and relative standard deviation (RSD).

[0113] result

[0114] Figure 2The fragmentation pattern of the cbz-Lys precursor ion under collision induced dissociation (CID) is shown. The most intense peak at m / z 91 was selected as the product ion for detection.

[0115] Figure 3 Ion chromatograms of cbz-Lys and internal standard (IS) from the following are shown: (1) pure buffer solution as a control; (2) enzyme reaction products from DBS extract in a surrogate matrix including a mixture of red blood cells and BSA solution in an optimal volume ratio; (3) enzyme reaction products from DBS extract of real whole blood samples from healthy individuals. cbz-Lys and IS show reproducible retention from different sample sources. In addition, the cbz-Lys peak intensity of the pure solution and the surrogate blood matrix is ​​very similar, which means that there is very little residual fC1-INH in the surrogate blood matrix. On the other hand, the cbz-Lys intensity derived from healthy blood samples with normal fC1-INH is significantly lower, indicating the inhibition of C1s enzyme activity by the presence of fC1-INH. This figure demonstrates the feasibility of detecting cbz-Lys after the enzymatic reaction of DBS extract.

[0116] Figure 4 The calibration curve for fC1-INH measurement in DBS is shown.

[0117] To evaluate the inter-run reproducibility of the DBS-based LC-MS / MS fC1-INH assay, pooled blood samples from six individual subjects were collected and analyzed on three different days using the DBS-based LC-MS / MS fC1-INH assay. The assay results are shown in Table 1. The precision of the assay (%CV ~ 10.3) was very good.

[0118] Table 1. Inter-run reproducibility of DBS-based LC-MS / MS fC1-INH assay. Pooled whole blood (WB) was prepared from six groups of subjects.

[0119]

[0120] In order to evaluate the intra-day reproducibility of the DBS-based LC-MS / MS fC1-INH assay, C1-INH was spiked into the red blood cell matrix at different concentrations as quality control (lower limit of quantification (LLOQ), low, medium and high). The samples were then analyzed using the DBS-based LC-MS / MS fC1-INH assay. The results are shown in Table 2. The fC1-INH levels obtained from the assay corresponded to the fC1-INH levels spiked into the sample, indicating the accuracy of the assay when measuring fC1-INH using DBS.

[0121] Table 2. Intra-day reproducibility of DBS-based LC-MS / MS fC1-INH assay

[0122]

[0123] Example 2 ： fC1-INH assay using DBS-based LC-MS / MS

[0124] This article describes the development and validation of a novel assay capable of measuring fC1-INH activity in DBS to diagnose HAE patients. The assay was validated in accordance with regulatory guidelines and industry best practices. DBS samples from HAE patients showed significantly lower fC1-INH activity, allowing HAE patients to be distinguished from healthy subjects.

[0125] Experimental Procedure

[0126] (i) Materials

[0127] N α -Benzyloxycarbonyl-Lys-thiobenzyl ester hydrochloride (Z-Lys-SBzl·HCl, C1s substrate) and N ε -Benzyloxycarbonyl-L-lysine-2,6,6-d3 (Nε-CBZ-L-lysine-d3, internal standard) were purchased from BACHEM (Torrance, California, USA) and CDN Isotopes (Quebec, Canada), respectively. Recombinant human complement component C1s (C1s) was purchased from R&D Systems (Minneapolis, MN, USA). Recombinant C1-INH was obtained in-house. Bovine serum albumin (BSA) was obtained from Americanbio (Natick, MA, USA). Sodium dodecyl sulfate (SDS) solution (10%) was a product of Sigma (St. Louis, MO, USA). 500 μL 96-well plates (Eppendorf Protein LoBind) were purchased from Eppendorf (Hamburg, Germany). 3 mm punches and Whatman #903 protein preservation cards were purchased from GE Health Care LifeScience (Little Chalfont, Buckinghamshire, UK). Desiccant (1 g) and biohazard bags were obtained from VWR (Radnor, PA, US). All other chemicals used in the study were of the highest grade and used without further purification.

[0128] (ii) Solution preparation

[0129] C1s working solution (0.5 μg / mL) was freshly prepared by diluting C1s stock solution (367 μg / mL) 734-fold using 0.5% BSA solution prepared in PBS buffer. Substrate stock solution was prepared by dissolving Z-Lys-SBzl·HCl in DMSO to a final concentration of 10 mM. Internal standard (IS) stock solution (2.5 mM) was prepared by dissolving Nε-CBZ-L-lysine-d3 in 5 mM Na2CO3 in methanol / water (50 / 50). Substrate, C1s and IS stock solutions were stored at -80°C before use. A substrate-IS cocktail containing 0.83 mM Z-Lys-SBzl·HCl and 33.3 μM Nε-CBZ-L-lysine-d3 was freshly prepared in PBS buffer. SDS solution (0.1%) was prepared by diluting 10% SDS 100-fold with methanol / water (80 / 20, V / V) solution.

[0130] (iii) Preparation of DBS samples

[0131] Whole blood from 24 patients previously diagnosed with HAE was provided with written informed consent from the patients. Blood was drawn into Vacutainer EDTA tubes and stored at 4°C. Within 24 hours of collection, the tubes were inverted several times to resuspend the blood cells, and 60 μL aliquots were spotted on filter paper spots. Normal human whole blood from 103 healthy subjects was purchased from BIOIVT (Westbury, NY, USA). Blood spots were dried at room temperature for at least 3 hours and stored at -20°C in sealed biohazard bags, each containing a desiccant.

[0132] (iv) Preparation of C1-INH-free blood as a surrogate matrix

[0133] In order to obtain a substitute matrix for the depletion of fCl-INH for the preparation of calibration curves and quality control (QC), six different batches of human whole blood samples (three men and three women, each 10mL) were prepared as described in Example 1. In short, whole blood was centrifuged at room temperature for 10 minutes at 1200x g. Supernatant plasma was removed, and red blood cells were washed with 5mL PBS solution to remove residual plasma and fCl-INH. Red blood cells were separated again by centrifugation at room temperature for 10 minutes at 1200xg. The obtained red blood cells without fCl-INH were merged. Alternative blood matrix was prepared by mixing the red blood cells of equal volume and 4.3% BSA prepared in PBS buffer.

[0134] (v) Preparation of calibrators and QC samples in DBS

[0135] By adding different concentrations of fC1-INH Calibration standards (calibrators) are prepared by mixing into alternative matrices. Nominal fC1-INH concentrations are 100, 200, 300, 500, 1000 and 1500 mU / mL, respectively. Quality control (QC) samples are prepared in alternative matrices or pooled human whole blood. Lower limit of quantitation (LLOQ) QC (100 mU / mL) and low QC (150 mU / mL) are prepared in alternative matrices. Considering endogenous C1-INH levels (EL), all other QCs are prepared in pooled whole blood: low-medium QC (EL), medium QC (EL+200 mU / mL), medium-high QC (EL+500 mU / mL) and high QC (EL+800 mU / mL). The preparation of calibrators and QCs in DBS follows the same procedure as above.

[0136] (vi) Sample extraction and enzymatic reaction

[0137] Samples were extracted and enzymatic reactions were performed as described in Example 1. In brief, DBS disks were cut using a 3 mm punch and transferred to 500 μL 96-well plates. Proteins in the disks were extracted by incubating in a Thermomixer incubator at a temperature of 37 ° C for 3 hours in 100 μL 0.5% BSA in PBS at a vortex of 1250 rpm. The plate was centrifuged at 4000 rpm for 3 minutes, and then 20 μL of the extract was transferred to another 96-well plate containing 50 μL C1s working solution. Then incubated at 800 rpm and 37 ° C for 1.5 hours. Subsequently, 105 μL of substrate-internal standard (substrate-IS) mixture was added to the above solution, and then incubated in the dark at 800 rpm for 40 minutes at room temperature. The reaction was terminated by transferring 50 μL of solution to 450 μL 0.1% SDS in methanol / water (80 / 20, v / v). Prior to LC-MS / MS analysis, the resulting samples were further diluted 200-fold with MeOH / water (80 / 20, v / v).

[0138] (vii) LC-MS / MS analysis

[0139] Reaction samples were analyzed by LC-MS / MS as described in Example 1. Briefly, the substrate (Z-Lys-SBzl·HCl), analyte (N α -benzyloxycarbonyl-L-lysine, cbz-Lys) and IS(N ε-CBZ-L-lysine-d3) was separated using a reverse phase column (Waters Xbridge Protein BEH C4, 3.5 μm, 2.1×50 mm) maintained at 30°C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The flow rate was 0.3 mL / min, and the mobile phase gradient was as follows (time, % B): 0 min, 4%; 0.5 min, 4%; 3.5 min, 12%; 3.6 min, 70%; 4.6 min, 70%; 4.7 min, 4%; 5.5 min, 4%. The autosampler was set at 4°C, and 3.0 μL of sample was injected using the partial-loop LC injection mode. Analytes and IS were quantified on an AB Sciex QTrap 6500 mass spectrometer operating in the selected reaction monitoring (SRM) mode using positive ions. The optimal mass spectrometry parameters were as follows: curtain gas, 20; collision gas, medium; ion spray voltage, 4000; temperature, 550; ion source gas 1, 50; ion source gas 2, 50; declustering voltage, 120; entrance voltage, 10.0; collision energy, 26.0; collision cell exit voltage, 12.0. Analytes and IS were monitored using SRM transitions from m / z 281.2 to m / z 91.0 and m / z 284.2 to m / z 91.0, respectively.

[0140] (viii) Validation of fitness for purpose measurements

[0141] The fit-for-purpose assay validation followed industry best practices for biomarker and DBS-based diagnostic assays (1-6). The method was validated for calibration curves, accuracy, precision, matrix effects, hematocrit, extraction, sampling location, and stability under a variety of conditions.

[0142] Accuracy (relative error or %RE) and precision (%RSD) were assessed by analyzing six replicates of QC samples in both surrogate and authentic matrices at each concentration level each day for three days. Intra-day and inter-day average accuracy and precision were calculated sequentially. Analyte carry-over was checked by injecting blank solutions after running the upper limit of quantitation (ULOQ) calibrators.

[0143] The effect of hematocrit level on assay performance was evaluated using whole blood from four healthy subjects. Briefly, plasma was separated from red blood cells by centrifugation at 1200 × g for 10 minutes. Then, different volumes of plasma and red blood cells were mixed to achieve hematocrit levels of 25%, 45%, 60%, and 75%, respectively. The DBS sample from 45% hematocrit was used as a reference for normalization of C1-INH activity at other hematocrit levels.

[0144] The effect of punch position on DBS spots and DBS spot variation was evaluated by using low and mid-high QC samples from one spot and 3-6 punch positions on a total of four different DBS spots ( Fig. 9 , Table 3). Low QCs were prepared in alternative matrix and mid-high QCs were prepared in pooled whole blood.

[0145] Table 3. Summary of DBS homogeneity tests

[0146]

[0147] Extraction efficiency was evaluated by comparing the results of pre-spiked and post-spiked samples using QCs prepared at 100, 600, and 1000 mU / mL in the surrogate matrix. Pre-spiked samples were prepared by spiking C1-INH into the surrogate matrix, and then DBS cards were prepared. On the other hand, post-spiked samples were prepared by spiking the same concentration of C1-INH (3.3 μL, corresponding to the wet blood volume in the 3.0 mm punch) into DBS extracts derived from blank surrogate matrix.

[0148] Whole blood stability of C1-INH activity was tested using equal volumes of pooled whole blood from six healthy subjects (3 males and 3 females). Before being added to the Whatman#903 protein preservation card, the samples were stored for up to 7 days at 4°C. Similarly, DBS samples prepared from pooled whole blood were stored at 45°C for 3 days and at room temperature for 134 days to assess stability under transport and storage conditions.

[0149] (ix) Data analysis

[0150] Data analysis was performed as described in Example 1. In brief, the peak area ratio of the analyte to the internal standard was determined by ABSciex Analyst (1.6.3). A 4-parameter logistic calibration curve was constructed using SoftMax Pro 7.0 with the peak area ratio and the spiked C1-INH concentration. Sample concentrations were calculated based on the following equation:

[0151]

[0152] Where y is the peak area ratio; x is the sample concentration; A, B, C and D are curve fitting parameters. In addition, Excel was used to calculate the mean, accuracy (%RE), standard deviation and relative standard deviation (%RSD).

[0153] result

[0154] Overall strategy

[0155] like Figure 5As shown, an LC-MS / MS assay was developed to measure the enzyme reaction product cbz-Lys in samples from dried blood spots. The assay consists of the following steps: (1) extraction of C1-INH from DBS cards; (2) binding of C1-INH to excess C1s; (3) reaction of unbound C1s with its substrate; (4) LC-MS / MS analysis of the enzyme reaction product cbz-Lys. The assay conditions, such as the relative concentrations of C1s and its substrate, the binding time and temperature between C1s and fC1-INH, the enzymatic reaction time and temperature between C1s and its substrate, and the LC-MS / MS conditions, were optimized during the pre-validation run.

[0156] Calibration matrix and calibration curve

[0157] C1-INH depleted blood matrix was prepared from pooled human whole blood to measure C1-INH activity in HAE patients, where C1-INH activity was lower than normal controls. C1-INH is a circulating protein and will remain in the plasma supernatant after centrifugation to spin down the red blood cells. The red blood cells were then supplemented with BSA solution to simulate the plasma protein concentration in real whole blood. 4.3% BSA (43 mg / mL) was mixed with the same volume of red blood cells to produce a viscosity similar to that of real whole blood, as shown by the same spot size in the filter paper. Fig. 6A and 6B As shown, the same analyte signals from the surrogate matrix and blank control confirmed the complete depletion of C1-INH, which means that there is no fC1-INH activity in the surrogate matrix. In contrast, in confluent healthy blood ( Figure 6C ) and pooled healthy blood spiked with 500 mU / mL of C1-INH ( Fig.6D ) showed a C1-INH-dependent signal reduction.

[0158] Figure 7 A typical calibration curve is shown by plotting the analyte signal versus six different C1-INH concentrations (mU / mL) spiked into the surrogate matrix using a four-parameter logistic curve fit. The curve shows a feasible range of 100 to 1500 mU / mL. The assay validation results showed that the six-point calibration curve met the pre-set criteria: (1) the relative error (%RE) of at least half of the calibrators at each concentration level should be ≤20%, with the exception of the LLOQ and ULOQ calibrators, which were ≤25%; (2) ≥75% of the total number of calibrators must be included in the calibration curve; and (3) no more than two consecutive validation runs failed. In 13 of the 14 passing analytical runs, the average inter-run accuracy of all calibrators ranged from -1.4% to 5.9% (Table 4).

[0159]

[0160]

[0161]

[0162] Determination precision and accuracy

[0163] The QC samples prepared in both surrogate matrix and real whole blood were used to evaluate the determination precision and accuracy.LLOQQC and low QC were prepared by mixing fCl-INH into the surrogate matrix to reach a final concentration of 100mU / mL and 150mU / mL respectively. The whole blood that converged was used as medium-low QC, and medium, medium-high and high QC samples were prepared by mixing fCl-INH into the whole blood that converged. Their nominal concentration is the sum of the average endogenous C1-INH concentration (518mU / mL, n=18) and the fCl-INH concentration that is mixed. As shown in Table 5, the intraday precision and accuracy range distribution is 4.4% to 11.6% and -11.1% to -2.1%, and the interday precision and accuracy are 8.1% to 13.1% and -10.3% to 0.9% respectively.

[0164] Table 5. Summary of intra-day and inter-day precision and accuracy

[0165]

[0166] Effect of hematocrit level

[0167] It is well documented that the hematocrit level of whole blood affects the dilution of blood in the filter paper and thus affects the DBS spot size, leading to assay bias (1-3, 6, 7). As the hematocrit level increases, the spot area of ​​the DBS sample decreases. Depending on sex and age, the hematocrit level in whole blood ranges from 28% to 67% (7). The effect of hematocrit on assay performance was studied at four hematocrit levels (25%, 45%, 60%, and 75%), with a 45% hematocrit level used as a control. fC1-INH activity was linearly correlated with the hematocrit level in the dried blood spot (Table 6). Hematocrit levels between 30%-60% had minimal effect on fC1-INH activity measurements (RE < 20%).

[0168] Table 6. Effect of hematocrit level on fC1-INH activity measured in DBS

[0169]

[0170] Punch sites for C1-INH activity

[0171] Another issue in DBS-based assays is the effect of the punch hole position on analyte measurement (1, 6, 8). To examine the effect of the punch hole position on the DBS card on C1-INH activity, a total of 18 replicate samples from the central and peripheral holes were collected from four points in the same subject, and low QC and mid-high QC samples were analyzed. Both precision and accuracy were within 15%, which means that the punch hole position does not affect the C1-INH activity measurement (Table 4).

[0172] Extraction efficiency

[0173] The extraction efficiency of C1-INH was evaluated using QC prepared in an alternative matrix. At QC concentrations of 100, 600, and 1000 mU / mL, the average extraction efficiency was 48.8%, 65.9%, and 58.2%, respectively (Table 7). During the sample testing process, a calibration curve was used for each run, and changes in the extraction efficiency did not affect the accuracy of the assay.

[0174] Table 7. Extraction efficiency of C1-INH in DBS samples

[0175] Concentration (mU / mL) Extraction efficiency ± SD (%, n = 4) RSD(%) 100 48.8±4.92 10.1 600 65.9±3.93 6.0 1000 58.2±3.45 5.9

[0176] Stability of fC1-INH in whole blood and DBS

[0177] The stability of fC1-INH was evaluated in whole blood. Previous reports have shown that fC1-INH is stable in patients and healthy subjects at room temperature for up to three days (9). After storage at 4°C for 7 days, fC1-INH activity in pooled healthy blood changed little (Table 8). To test the stability of fC1-INH in DBS, QC controls were placed in sealed bags with desiccant packs at room temperature and 45°C. C1-INH activity was measured in different punches on each card and showed minimal loss (<15%) after storage at 45°C for 3 days and at room temperature for 134 days (Table 8). These results indicate that fC1-INH can be transported and stored at ambient temperature in DBS without losing activity.

[0178] Table 8. Stability of fC1-INH activity in whole blood and DBS stored at different temperatures

[0179]

[0180]

[0181] The processed samples were placed in the autosampler at 4°C for two days before being injected to evaluate the stability of the injection. The results showed that the precision and accuracy met the predetermined acceptance criteria (Table 9). In addition, no analyte carryover was detected (data not shown).

[0182] Table 9. Summary of reinjection stability testing

[0183]

[0184] Analysis of DBS samples from healthy and HAE subjects

[0185] Using a validated assay, fC1-INH activity was measured in DBS samples collected from 103 healthy subjects and 24 HAE patients (9 males and 15 females). Figure 8 As shown. For the healthy subject group, fC1-INH activity ranged from 311 to 1090 mU / mL, with a mean activity and standard deviation (SD) of 573 and 135 mU / mL, respectively. Nevertheless, the C1-INH activity of all HAE subjects tested was below the LLOQ (100 mU / mL), except for one subject with a C1-INH of 158 mU / mL. A cut-off value of 303 mU / mL (mean - 2xSD) was established, which was able to fully distinguish between healthy and HAE subjects.

[0186] discuss

[0187] C1-INH serves to regulate a variety of inhibitory biological activities, including complement, contact, coagulation, and fibrinolytic systems (10-12). As a key inhibitor of three enzymes in the kallikrein-kinin cascade (factor XIIa, factor XIIf, and plasma kallikrein), normal fC1-INH levels prevent excessive production of bradykinin, a proinflammatory mediator that induces HAE attacks. Deficiency in fC1-INH activity leads to recurrent activation of the contact system, producing excess plasma kallikrein (pKal), an active proteolytic enzyme that in turn cleaves high molecular weight kininogen (HMWK) to release bradykinin (13-15). To date, more than 450 mutations have been identified throughout the C1-INH SERPING1 gene, and many are associated with fC1-INH deficiency in HAE patients (16).

[0188] Although there are different types of HAE, all are characterized by defective C1-INH function. Typically, HAE patients have fC1-INH levels between 5 and 30% of normal values, and diminished C1-INH activity is used as the most important laboratory parameter for HAE diagnosis (9). Conventional assays for fC1-INH activity measure fC1-INH activity indirectly through the reaction between C1s and its artificial substrate, Z-Lys-SBzl·HCl, to produce cbz-Lys and thiomethylbenzene. After derivatization with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), thiomethylbenzene is measured by a chromogenic assay (9). The presence of endogenous or spiked C1-INH inhibits the enzyme reaction, and therefore, the formation of the reaction product is inversely proportional to the C1-INH activity. Although conventional assays can effectively distinguish between healthy and HAE subjects in plasma or serum samples, their use in DBS is limited due to assay interference from red blood cells in whole blood. LC-MS / MS assays have unique advantages over conventional chromogenic assays. First, the chromogenic assay is based on two reaction steps, while the LC-MS / MS assay measures the direct product of the C1s reaction, so the assay variation can be lower. Second, the use of an internal standard (IS) in the assay also helps to correct for analytical biases during sample preparation and analysis. Third, calibration curves and QCs prepared in a surrogate matrix without C1-INH are included in each run to further enhance the accuracy and reproducibility of the assay. Considering the wide range of C1-INH activity in healthy subjects, the limited variation in C1-INH activity in HAE patients (5-30% of normal activity), and the narrow range of the enzyme kinetic reaction, minimizing assay variation can be a key factor in accurately measuring C1-INH activity.

[0189] In enzyme activity assays, calibration curves for the analyte are usually prepared in pure buffer solutions rather than in the authentic matrix, and the assay performance is monitored using QC samples. Although this approach is simple, it cannot be easily applied to C1-INH activity measurements because the enzyme reaction proceeds so rapidly that even slight changes in reaction conditions can affect the analytical results and, therefore, the accurate diagnosis of the patient. The concept of “surrogate matrix” was adopted in these studies, which has been widely used for the preparation of calibrators and QCs in biomarker assays because the authentic matrix contains endogenous analytes (15). The surrogate matrix should remove the analyte but be close to the authentic matrix in terms of digestion efficiency, ionization effect, and extraction yield. The use of a surrogate matrix derived from whole blood to prepare calibration curves and QCs is essential for accurately assessing the enzyme reaction in patient samples with low C1-INH activity. Using this approach, assays in the range of 100 to 1500 mU / mL were achieved. In addition, excellent accuracy and precision were achieved for both the surrogate matrix and the QC samples prepared from authentic whole blood, demonstrating the parallelism of the two matrices under the assay conditions.

[0190] The LC-MS / MS assay described here showed average intra- and inter-day variability of less than 15%, with negligible carryover between samples. Furthermore, C1-INH activity measurements were independent of the location of the punch in the DBS, which is not the case for some other analytes in the DBS (1). However, assay accuracy may be affected by hematocrit levels, and test results from samples with hematocrit levels below 30% or above 60% should be interpreted with caution.

[0191] It is important that analyte levels remain stable in DBS during sample transport and storage. C1-INH in whole blood is stable for 7 days at 4°C. In DBS, it is stable for up to 3 days when stored at 45°C and for 134 days when stored at room temperature. This is more than adequate for the entire duration of sample collection, transport to a central laboratory, and laboratory testing and retesting, even in developing countries.

[0192] C1-INH activity measured in 103 healthy subjects was normally distributed with a mean activity of 573 mU / mL and a standard deviation of 135 mU / mL. Among 24 HAE patients, 23 showed C1-INH activity <100 mU / mL, while the HAE sample with the highest C1-INH activity was 158 mU / mL, corresponding to 27.6% of the mean normal activity. The data support a clear distinction between samples from healthy individuals and corresponding samples from HAE patients.

[0193] In conclusion, described herein is a reliable assay for fC1-INH activity in DBS samples. This assay provides excellent reproducibility and accuracy for the diagnosis of HAE patients. Simple sample collection and extended shipping and storage stability expand the availability of diagnostic testing, especially in settings with limited or no access to well-equipped clinical laboratories. This assay shows great potential to fundamentally change the diagnostic algorithm for HAE worldwide.

[0194] refer to

[0195] 1. Holub M, Tuschl K, Ratschmann R, Strnadova KA, Muhl A, Heinze G, et al., Influence of hematocrit and localization of punch in dried blood spots onlevels of amino acids and acylcarnitines measured by tandem massspectrometry. Clin Chim Acta 2006; 373:27-31.

[0196] 2. De Jesus VR, Zhang XK, Keutzer J, Bodamer OA, Muhl A, Orsini JJ, et al. Development and evaluation of quality control dried blood spot materials in newborn screening for lysosomal storage disorders. Clin Chem 2009;55:158 - 64.

[0197] 3. de Vries R, Barfield M, van de Merbel N, Schmid B, Siethoff C, Ortiz J, et al. The effect of hematocrit on bioanalysis of dbs: Results from the ebf dbs - microsampling consortium. Bioanalysis 2013;5:2147 - 60.

[0198] 4. Lee JW, Devanarayan V, Barrett YC, Weiner R, Allinson J, Fountain S, et al. Fit - for - purpose method development and validation for successful biomarker measurement. Pharm Res 2006;23:312 - 28.

[0199] 5. McDade TW. Development and validation of assay protocols for use with dried blood spot samples. Am J Hum Biol 2014;26:1 - 9.

[0200] 6. Timmerman P, White S, Globig S, Ludtke S, Brunet L, Smeraglia J. Ebf recommendation on the validation of bioanalytical methods for dried blood spots. Bioanalysis 2011;3:1567 - 75.

[0201] 7. Denniff P, Spooner N. The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. Bioanalysis 2010; 2: 1385 - 95.

[0202] 8. Cobb Z, de Vries R, Spooner N, Williams S, Staelens L, Doig M, et al. In-depth study of homogeneity in DBS using two different techniques: Results from the EBF DBS-microsampling consortium. Bioanalysis 2013; 5: 2161 - 9.

[0203] 9. Wagenaar-Bos IG, Drouet C, Aygoren-Pursun E, Bork K, Bucher C, Bygum A, et al. Functional C1-inhibitor diagnostics in hereditary angioedema: Assay evaluation and recommendations. J Immunol Methods 2008; 338: 14 - 20.

[0204] 10. Bork K, Davis-Lorton M. Overview of hereditary angioedema caused by C1-inhibitor deficiency: Assessment and clinical management. Eur Ann Allergy Clin Immunol 2013; 45: 7 - 16.

[0205] 11. Csuka D, Veszeli N, Varga L, Prohaszka Z, Farkas H. The role of the complement system in hereditary angioedema. Mol Immunol 2017; 89: 59 - 68.

[0206] 12.Ratnoff OD、Pensky J、Ogston D、Naff GB.The inhibition of plasmin,plasma kallikrein,plasma permeability factor,and the C'1r subcomponent of thefirst component of complement by serum C'1esterase inhibitor.J Exp Med 1969;129:315-31.

[0207] 13.Nzeako UC、Frigas E、Tremaine WJ.Hereditary angioedema:A broadreview for clinicians.Arch Intern Med 2001;161:2417-29.

[0208] 14.Bernstein JA、Moellman J.Emerging concepts in the diagnosis andtreatment of patients with undifferentiated angioedema.Int J Emerg Med 2012;5:39.

[0209] 15.Zhang G、Sexton DJ、Faucette RR、Qiu Y,Wu J.2d-lc-ms / ms to measurecleaved high-molecular-weight kininogen in human plasma as a biomarker forC1-INH-HAE.Bioanalysis 2017;9:1477-91.

[0210] 16. Johnsrud I, Kulseth MA, Rodningen OK, Landro L, Helsing P, Waage Nielsen E, Heimdal K. A nationwide study of norwegian patients with hereditary angioedema with C1 inhibitor deficiency identified six novel mutations in SERPING1. PLoS One 2015;10:e0131637.

[0211] 17. Maurer M, Magerl M, Ansotegui I, Aygoren-Pursun E, Betschel S, Bork K et al., The international wao / eaaci guideline for the management of hereditary angioedema - the 2017 revision and update. Allergy 2018;73:1575 - 96.

[0212] 18. Li HH, Busse P, Lumry WR, Frazer-Abel A, Levy H, Steele T et al., Comparison of chromogenic and ELISA functional C1 inhibitor tests in diagnosing hereditary angioedema. J Allergy Clin Immunol Pract 2015;3:200 - 5.

[0213] 19. Ganz N, Singrasa M, Nicolas L, Gutierrez M, Dingemanse J, Dobelin W, Glinski M. Development and validation of a fully automated online human dried blood spot analysis of bosentan and its metabolites using the sample card and prep dbs system. J Chromatogr B Analyt Technol Biomed Life Sci 2012;885 - 886:50 - 60.

[0214] 20. Holub M, Tuschl K, Ratschmann R, Strnadova KA, Muhl A, Heinze G et al. Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry. Clin Chim Acta 2006;373:27 - 31.

[0215] 21. Matern D, Oglesbee D, Tortorelli S. Newborn screening for lysosomal storage disorders and other neuronopathic conditions. Dev Disabil Res Rev 2013;17:247 - 53.

[0216] 22. Mokhtariye A, Hagh - Nazari L, Varasteh AR, Keyfi F. Diagnostic methods for lysosomal storage disease. Rep Biochem Mol Biol 2019;7:119 - 28.

[0217] 23. Zhang XK, Elbin CS, Chuang WL, Cooper SK, Marashio CA, Beauregard C, Keutzer JM. Multiplex enzyme assay screening of dried blood spots for lysosomal storage disorders by using tandem mass spectrometry. Clin Chem 2008;54:1725-8.

[0218] 24. Olivova P, van der Veen K, Cullen E, Rose M, Zhang XK, Sims KB, et al., Effect of sample collection on alpha-galactosidase an enzyme activity measurements in dried blood spots on filter paper. Clin Chim Acta 2009; 403:159-62.

[0219] Other Implementations

[0220] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0221] According to the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from its spirit and scope, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0222] Equivalent content

[0223] Although several inventive embodiments have been described and illustrated herein, a person of ordinary skill in the art will readily conceive of various other ways and / or structures for performing functions and / or obtaining results and / or one or more advantages described herein, and each of these changes and / or variations is considered to be within the scope of the inventive embodiments described herein. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize that, or will be able to determine many equivalents of the specific inventive embodiments described herein using only routine experiments. Therefore, it should be understood that the foregoing embodiments are given by way of example only, and within the scope of the appended claims and their equivalents, the inventive embodiments may be implemented in a manner different from that specifically described and claimed. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, if these features, systems, articles, materials, kits, and / or methods are consistent with each other, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention disclosed herein.

[0224] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0225] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to each subject matter cited, in some cases in their entirety.

[0226] Unless there is a clear indication to the contrary, the indefinite articles "a" and "an" used herein in the specification and claims should be understood to mean "at least one / kind". The phrase "and / or" as used herein in the specification and claims should be understood to mean "one or two" of the elements so combined, i.e., jointly existing in some cases and separately existing in other cases. Multiple elements listed with "and / or" should be interpreted in the same way, i.e., "one or more" of the elements so connected. Other elements may optionally exist, in addition to the elements clearly identified by the "and / or" clause, regardless of whether they are related or unrelated to those specifically identified. Therefore, as a non-limiting example, when used in combination with open language such as "comprising", mentioning "A and / or B" can refer only to A (optionally including other elements other than B) in one embodiment; in another embodiment, only to B (optionally including elements other than A); in another embodiment, it is related to A and B (optionally including other elements), etc.

[0227] As used in this specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of a number or a list of elements, but also including more than one of the many or a list of elements, and optionally other unlisted items. Only terms that clearly indicate the opposite, such as "only one" or "exactly one" or when used in the claims, "consisting of...", will refer to the inclusion of exactly one of the many or a list of elements. Generally, the term "or" used herein should only be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both") before an exclusive term (e.g., "one of the two", "one of them", "only one" or "exactly one"). When used in the claims, "essentially consisting of..." should have its ordinary meaning used in the field of patent law.

[0228] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to refer to at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. In addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, the definition also allows that elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") may refer to at least one, optionally including more than one A, without B (and optionally including elements other than B) in one embodiment; to at least one, optionally including more than one B, without A (and optionally including elements other than A) in another embodiment; to at least one, optionally including more than one A and at least one, optionally including more than one B (and optionally including other elements) in another embodiment, and so on.

[0229] It should also be understood that, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order of the steps or acts of the method as described unless otherwise indicated to the contrary.

Claims

1. Use of a support member for spotting a blood sample and a reagent for measuring the level of functional C1-esterase inhibitor (fC1-INH) in a sample in the preparation of a diagnostic agent for diagnosing a subject at risk of or suffering from a C1-INH deficiency-mediated disorder, wherein the diagnosis comprises: (i) spotting a blood sample from a subject on a support member; (ii) drying the blood sample on the support member to form a dried blood spot; (iii) extracting protein from the dried blood spot of (ii); and (iv) measuring the level of fC1-INH in the protein extracted in (iii), if present; Measurement of fC1-INH levels includes: (a) incubating the extracted protein with complement component 1s (C1s) and a C1s substrate, in an optional order, to produce a C1s substrate product; (b) measuring the level of the C1s substrate product produced in step (a); and (c) determining the level of fC1-INH in the dried blood spot based on the level of C1s substrate product measured in step (b).

2. The use according to claim 1, wherein step (a) is performed by incubating the extracted protein with C1s and a C1s substrate to produce a reaction mixture.

3. The method according to claim 1 or claim 2, wherein the measuring step of step (b) is performed by liquid chromatography-mass spectrometry.

4. The use according to claim 1 or claim 2, wherein the C1s substrate is N α -benzoyl-Lys-thiobenzyl ester and the C1s substrate product is N α -benzyloxycarbonyl-L-lysine (cbz-Lys).

5. The use according to claim 1 or claim 2, wherein the extraction of (iii) is performed by incubating the dried blood spot with bovine serum albumin (BSA) in PBS buffer for at least 3 hours.

6. The use according to claim 1 or claim 2, wherein the support member is filter paper.

7. The method according to claim 1 or claim 2, wherein the drying in step (ii) is carried out at room temperature for at least 3 hours.

8. The use according to claim 1 or claim 2, further comprising obtaining the blood sample from the subject.

9. The use according to claim 1 or claim 2, wherein the blood sample is a whole blood sample.

10. The use according to claim 1 or claim 2, wherein the subject is a human subject.

11. The use according to claim 10, wherein the subject suffers from, is suspected of suffering from, or is at risk of suffering from hereditary angioedema (HAE).

12. The use according to claim 11, wherein the HAE is type I HAE or type II HAE.

13. The use of claim 1 or claim 2, further comprising determining whether the subject suffers from a C1-INH deficiency-mediated disorder, wherein a reduced level of fC1-INH product compared to a control indicates that the subject suffers from a C1-INH deficiency-mediated disorder.

14. The use of claim 1 or claim 2, further comprising identifying appropriate treatment for the subject based on the level of fC1-INH.

15. The use of claim 1 or claim 2, further comprising identifying the subject as a candidate for treatment of the C1-INH deficiency-mediated disorder based on the level of fC1-INH determined in step (c).

16. The use of claim 1 or claim 2, further comprising administering a therapeutic agent to the subject if the subject is identified as being at risk for or suffering from a C1-INH deficiency-mediated disorder.

17. The use according to claim 16, wherein the therapeutic agent is a plasma kallikrein (pKal) inhibitor, a bradykinin B2 receptor antagonist or a C1 esterase inhibitor.

18. The use according to claim 16, wherein the therapeutic agent is ecallantide, lanarumab, icatibant or a human plasma-derived C1 esterase inhibitor.

19. The use according to claim 13, wherein the C1-INH deficiency-mediated disorder is HAE.

20. The use according to claim 19, wherein the HAE is type I HAE or type II HAE.

Images