Methods, liposome collections, and kits for determining complement activity

Abstract

The present invention relates to methods for determining complement activity, including determining classical complement pathway activity, determining alternative complement pathway activity, and / or determining lectin complement pathway activity. The present invention further relates to a liposome collection for determining complement activity. The present invention further relates to a kit for determining complement activity. The present invention also relates to a liposome collection or kit for use in methods for diagnosing, monitoring, or treating complement-associated disorders.

Description

[Technical Field]

[0001] The present invention relates to a method for determining complement activity. The present invention further relates to a liposome collection for determining complement activity. The present invention further relates to a kit for determining complement activity. The present invention also relates to a liposome collection or kit for use in a method for diagnosing, monitoring, or treating a complement-associated disorder. [Background technology]

[0002] The complement system is a part of the immune system that plays a key role in fighting infections, but abnormalities in this system can lead to a variety of diseases. A fully functional and balanced complement system plays a role in the physiology of a healthy organism and is a multidimensional innate immune surveillance system. On the one hand, it controls cell survival by removing apoptotic cells, and on the other hand, it inhibits the growth of microorganisms that can cause infectious diseases. The complement system involves the coordinated activity of over 40 proteins that are activated by at least three different stimuli: the classical pathway, the lectin pathway, and the alternative pathway. Once activated, it tags cells for phagocytosis, releases anaphylatoxins, and lyses the cells.

[0003] Complement-mediated responses in the body are affected by genetic defects, viral and microbial infections, autoimmune reactions, complement-targeting therapeutic agents, and artificial surfaces in hemodialysis and extracorporeal circuits. To date, complement activation ability has primarily been measured in standard clinical diagnostic laboratories to determine complement deficiencies or abnormalities in complement regulation in autoimmune reactions and used for patient diagnosis.

[0004] Current complement activation assays have significant limitations. They are not suitable for determining high levels of function or dysregulation. Furthermore, the assays are expensive due to multiple reagents, washing steps, and long assay times. A major drawback is the use of animal products, which results in a short shelf life and significant batch-to-batch variability. The characteristics of existing complement activation assays have not kept pace with rapid drug development. In 2020, of the vast number of 870 clinical studies evaluating various complement-targeting therapies involving nearly 2.43 million patients, only 16 studies assessed complement activation as an endpoint measure.

[0005] In recent years, a new class of immunosuppressive drugs that target the complement system has been approved for clinical use. These recent advances have created a demand for new tests, including blood tests, for the diagnosis, follow-up, and management of complement-related diseases and for monitoring treatment response. However, there is currently a significant shortage of blood tests and laboratory analyses of the complement system.

[0006] Therefore, there is a need for efficient complement activity assays, including methods and tools for determining complement activity. For example, there is a need for inexpensive, rapid, and high-throughput assays for complement activity. In particular, there is a need for means that can specifically determine complement activity, including the activity of the three complement pathways. There is also a need for means for treating, diagnosing, and monitoring complement-related disorders. Summary of the Invention

[0007] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any number in any way to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This specification should be understood to support and encompass embodiments combining two or more of the explicitly described embodiments, or combining one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context dictates otherwise, any permutation and combination of all elements described herein should be considered to be disclosed by the description of this application.

[0008] In a first aspect, the present invention provides a method of determining complement activity, comprising: A) determining classical complement pathway activity; B) determining alternative complement pathway activity; and / or C) determining lectin complement pathway activity; A) determining the classical complement pathway activity; a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol; Optionally, the one or more liposomes comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; b) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; c) detecting the released marker; Including, B) determining the alternative complement pathway activity; a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise an alternative complement pathway activator, preferably lipopolysaccharide, more preferably about 0.01 mol% to about 5 mol% lipopolysaccharide, even more preferably about 1 mol% lipopolysaccharide; Optionally, the one or more liposomes comprise cholesterol, preferably ≦10 mol % cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor; Preferably, the classical complement pathway inhibitor and / or the lectin complement pathway inhibitor comprises EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP, preferably EGTA and / or C1 inhibitor; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; Including, C) determining the lectin complement pathway activity; a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; The present invention relates to a method comprising:

[0009] In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) do not comprise an antibody or antigen-binding fragment thereof, such as an anti-2,4,6-trinitrophenyl (TNP) antibody, an anti-PEG antibody, or an antigen-binding fragment thereof. In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) are not liposomes activated by an antibody or antigen-binding fragment thereof, such as an anti-2,4,6-trinitrophenyl (TNP) antibody, an anti-PEG antibody, or an antigen-binding fragment thereof. In these embodiments, liposome-mediated classical pathway activation, alternative pathway activation, and / or lectin complement pathway activation is not mediated by an antibody or antigen-binding fragment thereof, such as an anti-2,4,6-trinitrophenyl (TNP) antibody, an anti-PEG antibody, or an antigen-binding fragment thereof.

[0010] In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) do not contain a ligand, such as a hapten ligand. In this embodiment, liposome-mediated classical pathway activation, alternative pathway activation, and / or lectin complement pathway activation do not occur via a ligand, such as a hapten ligand. In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) are not liposomes activated by a ligand, such as a hapten.

[0011] In one embodiment, liposome-induced classical pathway activation, alternative pathway activation, and / or lectin complement pathway activation is not mediated by TNP-DPPE, and in this embodiment, the liposomes provided in step A)a), the liposomes provided in step B)a), and / or the liposomes provided in step C)a) are not TNP-DPPE-induced liposomes.

[0012] In one embodiment, the classical complement pathway activator, alternative pathway activator, and / or lectin complement pathway activator is not an antibody or antigen-binding fragment thereof, such as an anti-2,4,6-trinitrophenyl (TNP) antibody, an anti-PEG antibody, or an antigen-binding fragment thereof, or a ligand, such as a hapten ligand.

[0013] In one embodiment, the one or more liposomes for determining classical complement pathway activity, alternative complement pathway activity, and / or lectin complement pathway activity do not comprise an antibody or antigen-binding fragment thereof, such as an anti-2,4,6-trinitrophenyl (TNP) antibody, an anti-PEG antibody, or an antigen-binding fragment thereof, or a ligand, such as a hapten ligand. In this embodiment, activation of the classical complement pathway, alternative complement pathway, and / or lectin complement pathway does not include lysis of the liposome induced by the antibody and / or ligand.

[0014] In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) are not PEGylated liposomes.

[0015] In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) do not comprise paragloboside.

[0016] In one embodiment, the A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity comprises: Optionally, detecting a basal level of the marker prior to the step of contacting the one or more liposomes with the sample at a time point ranging from about 1 second to about 20 minutes, preferably from about 1 second to about 15 minutes, after initial contact of the one or more liposomes with the sample, and / or using inactivated serum and / or buffer; Optionally, determining complement activity by comparing the released marker detected in steps A)c), B)d), and C)d), respectively, with the basal level of the marker detected in the step of detecting the basal level of the marker, Preferably, a higher level of the released marker detected in steps A) c), B) d), and C) d), respectively, compared to the basal level of the marker, is indicative of complement activity. Further includes:

[0017] In one embodiment, the buffer used in any of the steps of the method according to the first aspect of the invention is not and / or does not comprise gelatin veronal buffer (GVB). In this embodiment, the method has an advantage over methods that use gelatin veronal buffer (GVB) because GVB contains the toxic component barbital, which is not present in the buffer used in any of the steps of the method according to the first aspect of the invention.

[0018] In one embodiment, said detecting in step A)c), step B)d), and / or step C)d) comprises or consists of time-resolved detection of said released markers.

[0019] In one embodiment, said detecting in step A)c), step B)d), and / or step C)d) comprises or consists of time-resolved detection of said released marker, wherein an increase or decrease in said released marker over time, preferably an increase in said released marker over time, is indicative of complement activity.

[0020] In one embodiment, the marker is a fluorescent marker, preferably pyrenetetrasulfonic acid, sulforhodamine B, indocyanine green such as 2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, or carboxyfluorescein; an electrochemical marker, preferably hexafluor a colorimetric marker, preferably sulforhodamine B; a chemiluminescent marker, preferably luminol or a derivative of luminol such as mCOOH-luminol; an electrochemiluminescent marker, preferably luminol, a derivative of luminol, or ruthenium bipyridyl; a bioluminescent marker such as GFP, other bioluminescent proteins, or luciferase; and combinations thereof; preferably, the marker is water-soluble; Preferably, the marker is selected from a fluorescent marker, an electrochemical marker, and combinations thereof; More preferably, the marker is selected from sulforhodamine B and mCOOH-luminol.

[0021] In one embodiment, the marker is not carboxyfluorescein.

[0022] In one embodiment, the marker is a fluorescent marker, and the marker is quenched in the undissolved liposomes; Preferably, the one or more liposomes are non-dissolved liposomes prior to said dissolution of the one or more liposomes; Optionally, An increase in the released marker detected in step A)c), step B)d), and / or step C)d) over time indicates complement activity.

[0023] In one embodiment, the one or more liposomes comprising a marker provided in step A)a) comprise a first marker, the one or more liposomes comprising a marker provided in step B)a) comprise a second marker, and the one or more liposomes comprising a marker provided in step C)a) comprise a third marker, wherein the first marker, the second marker, and the third marker are different markers.

[0024] In one embodiment, the method comprises determining, preferably simultaneously or sequentially, at least two of: A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and C) determining lectin complement pathway activity; Preferably, the detection of said released markers in steps A)c), B)d), and / or C)d) is carried out simultaneously or sequentially, preferably simultaneously.

[0025] In one embodiment, the method comprises determining lectin complement pathway activity; Determining the lectin complement pathway activity includes: a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; Includes:

[0026] In one embodiment, the method comprises A) determining classical complement pathway activity and C) determining lectin complement pathway activity; A) Determining the classical complement pathway activity includes: a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol; Optionally, the one or more liposomes comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; b) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; c) detecting the released marker; Including, C) determining the lectin complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; Includes:

[0027] In one embodiment, the method comprises B) determining alternative complement pathway activity and C) determining lectin complement pathway activity; B) determining the alternative complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise an alternative complement pathway activator, preferably lipopolysaccharide, more preferably about 0.01 mol% to about 5 mol% lipopolysaccharide, even more preferably about 1 mol% lipopolysaccharide; Optionally, the one or more liposomes comprise cholesterol, preferably ≦10 mol % cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor; Preferably, the classical complement pathway inhibitor and / or the lectin complement pathway inhibitor comprises EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP, preferably EGTA and / or C1 inhibitor; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; Including, C) determining the lectin complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; Includes:

[0028] In one embodiment, the method comprises A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and C) determining lectin complement pathway activity; A) Determining the classical complement pathway activity includes: a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol; Optionally, the one or more liposomes comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; b) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; c) detecting the released marker; Including, B) determining the alternative complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise an alternative complement pathway activator, preferably lipopolysaccharide, more preferably about 0.01 mol% to about 5 mol% lipopolysaccharide, even more preferably about 1 mol% lipopolysaccharide; Optionally, the one or more liposomes comprise cholesterol, preferably ≦10 mol % cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor; Preferably, the classical complement pathway inhibitor and / or the lectin complement pathway inhibitor comprises EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP, preferably EGTA and / or C1 inhibitor; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; Including, C) determining the lectin complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes containing a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released marker; Includes:

[0029] In one embodiment, the one or more liposomes provided in step A)a) comprise cholesterol and phospholipids; preferably, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); more preferably, at least 30 mol % cholesterol, preferably at least 40 mol % cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 10 mol % to 30 mol %, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in the range of 25 mol % to 45 mol %; Optionally, the one or more liposomes provided in step A)a) further comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; the one or more liposomes provided in step B)a) comprise phospholipids and cholesterol, preferably ≦10 mol% cholesterol; preferably 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; more preferably 65 mol% to 85 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 10 mol% to 30 mol% 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; and / or The one or more liposomes provided in step C)a) comprise phospholipids and ≦10 mol% cholesterol; preferably, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; more preferably, 65 mol% to 85 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 10 mol% to 30 mol% of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; Optionally, the one or more liposomes provided in step A)a), step B)a), and / or step C)a) comprise 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine; Optionally, the one or more liposomes provided in step B)a) and / or the one or more liposomes provided in step C)a) comprise a classical complement pathway inhibitor, preferably an anti-C1q85 antibody or antigen-binding fragment thereof.

[0030] In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) are selected from anionic liposomes, PEGylated liposomes, and cationic liposomes, preferably anionic liposomes.

[0031] In one embodiment, step B)b) comprises the step of: 2+ The sample and / or the one or more liposomes are treated with Ca in the presence of at least 0.001 mM MgCl, preferably in the presence of at least 0.1 mM MgCl, even more preferably in the presence of at least 0.5 mM MgCl, for example about 1 mM MgCl. 2+ The method comprises contacting the compound with a complexing agent, preferably a classical complement pathway inhibitor selected from EGTA and / or a lectin complement pathway inhibitor.

[0032] In one embodiment, the method includes a positive control and / or a negative control; The positive control comprises contacting the one or more liposomes provided in steps A)a), B)a), and / or C)a) with a detergent and / or solvent, preferably a detergent, more preferably a detergent selected from n-octyl-β-d-glucoside, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polyoxyethylene(20) sorbitan monolaurate, and polyoxyethylene(80) sorbitan monooleate; The negative control comprises replacing the sample, optionally the serum sample, in steps A)a), B)a), and / or C)a) with a negative control sample comprising or consisting of inactive serum.

[0033] In a further aspect, the present invention provides a liposome collection for determining complement activity, preferably for A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity, comprising: - A) a liposome for determining classical complement pathway activity, comprising a marker, cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol, and a phospholipid; Preferably, the markers include cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); More preferably, the marker comprises at least 30 mol% cholesterol, preferably at least 40 mol% cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 10 mol% to 30 mol%, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) in the range of 25 mol% to 45 mol%; Optionally, the liposome further comprises a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; - B) a liposome for determining alternative complement pathway activity, comprising a marker, an alternative complement pathway activator, cholesterol, preferably ≦10 mol % cholesterol, and a phospholipid; Preferably, the markers include an alternative complement pathway activator, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); More preferably, the marker, alternative complement pathway activator, comprises ≦10 mol % cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 65 mol % to 85 mol %, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in the range of 10 mol % to 30 mol %; Preferably, the alternative complement pathway activator is a liposome comprising lipopolysaccharide, preferably from about 0.01 mol % to about 5 mol % lipopolysaccharide, more preferably about 1 mol % lipopolysaccharide; and / or - C) a liposome for determining lectin complement pathway activity, comprising a marker, a lectin complement pathway activator, cholesterol, preferably ≦10 mol % cholesterol, and a phospholipid; Preferably, the markers include lectin complement pathway activator, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); More preferably, the marker comprises a lectin complement pathway activator, ≦10 mol % cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 65 mol % to 85 mol %, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in the range of 10 mol % to 30 mol %; Preferably, the liposome wherein the lectin complement pathway activator comprises a sugar moiety, an acetyl moiety, and / or a sialic acid; Including, Optionally, the liposome for determining alternative complement pathway activity B) and / or the liposome for determining lectin complement pathway activity C) comprise a classical complement pathway inhibitor, preferably an anti-C1q85 antibody or antigen-binding fragment thereof; Preferably, the liposome collection comprises A) and B), A) and C), B) and C), or A), B) and C).

[0034] In one embodiment, the liposome collection is used in a method for determining complement activity as defined herein.

[0035] In a further aspect, the present invention provides a method for producing a composition comprising: - a liposome collection as defined herein; - a buffer, preferably a liposomal complement buffer; - optionally a classical complement pathway inhibitor, an alternative complement pathway inhibitor, and / or a lectin complement pathway inhibitor; preferably EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP; more preferably EGTA and / or C1 inhibitor; - optionally inactivated serum; preferably serum inactivated by EDTA, EGTA, heat, ultrasound and / or solvents such as water-soluble organic solvents; - optionally a surfactant, preferably n-octyl-β-d-glucoside; optionally an osmotic agent, preferably a sucrose solution; - optionally instructions for determining complement activity, preferably instructions for determining classical complement pathway activity, alternative complement pathway activity, and / or lectin complement pathway activity; more preferably instructions for a method of determining complement activity as defined herein. The present invention relates to a kit for determining complement activity, comprising:

[0036] In one embodiment, the kit is for use in a method for determining complement activity as defined herein.

[0037] In a further aspect, the present invention relates to a liposome collection as defined herein or a kit as defined herein for use in a method for diagnosing, monitoring or treating a complement-associated disorder, preferably a complement-associated disease; optionally, the complement-associated disease is selected from eye diseases, sepsis, kidney diseases, infectious diseases, primary complement gene deficiencies, and autoimmune diseases, and preferably selected from systemic lupus erythematosus, rheumatoid arthritis, hereditary angioedema, meningococcal disease, anti-factor H antibody-associated diseases, recurrent suppurative infections, hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, neuromyelitis optica disorder, atypical hemolytic uremic syndrome, and C3 glomerulopathy.

[0038] In one embodiment, the use in a method of diagnosing, monitoring, or treating a complement-associated disorder comprises performing a method of determining complement activity as defined herein.

[0039] In a further aspect, the present invention relates to a method for diagnosing, monitoring or treating a complement-associated disorder, preferably a complement-associated disease, comprising administering a liposome collection as defined herein or a kit as defined herein to a patient in need thereof and / or to a sample from a patient in need thereof.

[0040] In one embodiment, the complement-associated disease is selected from eye diseases, sepsis, kidney diseases, infectious diseases, primary complement gene deficiencies, and autoimmune diseases, and is preferably selected from systemic lupus erythematosus, rheumatoid arthritis, hereditary angioedema, meningococcal disease, anti-factor H antibody-associated diseases, recurrent suppurative infections, hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, neuromyelitis optica disorder, atypical hemolytic uremic syndrome, and C3 glomerulopathy.

[0041] In one embodiment, the administering comprises administering an effective amount of a liposome collection as defined herein and / or a kit as defined herein to a patient in need thereof and / or a sample from a patient in need thereof.

[0042] In one embodiment, the method of diagnosing, monitoring, or treating a complement-associated disorder comprises performing a method of determining complement activity as defined herein.

[0043] In a further aspect, the present invention relates to the use of the liposome collection as defined herein or the kit as defined herein for the manufacture of a medicament, preferably a medicament for diagnosing, monitoring or treating a complement-associated disorder, preferably a complement-associated disease as defined herein. [Brief explanation of the drawings]

[0044] The invention will now be further described by reference to the following drawings. All methods described in the following figure legends were carried out as detailed in the Examples. [Figure 1]Time-resolved fluorescence intensity of a complement assay performed on a mixture of anionic liposomes: AB-PTSA-liposomes (A, 100 mM PTSA encapsulated, antibody-modified) + CG-liposomes (B, 10 mM IR783 encapsulated, 1% LPS-modified) + AB-SRB-liposomes (C, 10 mM SRB encapsulated, antibody-modified). Complement assays were performed in liposome complement buffer containing a buffer control (black), activated serum (red), inactivated serum (blue), and an n-octyl-β-d-glucoside (OG) positive control (green). The serum content was 10% by volume per well. The serum source was IR35577 (i.e., Innovative Research human complement serum batch #35577). PTSA fluorescence: λEx = 375(5) nm and λEm = 405(5) nm, 37°C, gain = 150. CG fluorescence: λEx = 790(13) nm and λEm = 820(16) nm, 37°C, gain = 175. SRB fluorescence: λEx = 565(5) nm and λEm = 585(5) nm, 37°C, gain = 150, n = 3. [Figure 2] Normalized endpoint data (60 min) of time-resolved fluorescence intensity from a complement assay performed on a mixture of anionic liposomes: AB-PTSA-liposomes (100 mM PTSA encapsulated, antibody-modified) + CG-liposomes (10 mM IR783 encapsulated, 1% LPS-modified) + AB-SRB-liposomes (10 mM SRB encapsulated, antibody-modified). Normalized to the positive control (30 mM OG + 10% aS by volume) after 60 min. Yellow represents PTSA fluorescence, purple represents SRB fluorescence, and green represents CG fluorescence. Patterned bars represent data from single liposome control samples, while unpatterned bars represent the intensity of the liposome mixture. Complement assays were performed in liposome complement buffer. The serum content was 10% by volume per well. The serum source was IR35577. PTSA fluorescence: λEx = 375(5) nm and λEm = 405(5) nm, 37°C, gain = 150. CG fluorescence: λEx = 790(13) nm and λEm = 820(16) nm, 37°C, gain = 175. SRB fluorescence: λEx = 565(5) nm and λEm = 585(5) nm, 37°C, gain = 150, n = 3. [Figure 3] Normalized endpoint data (60 min) from a time-resolved complement assay performed on EGTA-inactivated anionic cholesterol-rich liposomes (42%, KH210127) are shown. 0.4 mM EGTA in LCB containing 1 mM MgCl2 was present in each well (except for the control) to inhibit the classical and lectin pathways. EGTA inactivation was performed without incubation prior to addition to the wells. After 60 min, normalization was performed against a positive control (30 mM OG + 10% aS, by volume). Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565(5) nm and λEm = 585(5) nm, 37°C, gain = 150, n = 3. As demonstrated, cholesterol-rich liposomes are induced via the classical and / or lectin pathways. This can be concluded from the complete inhibition of lysis of these liposomes in the presence of EGTA. If the alternative pathway were involved, some lysis would remain. The question of whether this type of liposome is specific to one complement pathway, the classical pathway or the lectin pathway, is answered by Figures 11-21, which show that cholesterol-rich liposomes are specifically induced via the classical pathway. [Figure 4]This figure shows a time-resolved complement assay performed on anionic cholesterol-rich liposomes (42%, KH210127) inactivated with EGTA. 0.4 mM EGTA in LCB containing 1 mM MgCl2 was present in each well (except for the control) to inhibit the classical and lectin pathways. EGTA inactivation was performed prior to addition to the wells without incubation. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565(5) nm and λEm = 585(5) nm, 37°C, gain = 150, n = 3. As shown, cholesterol-rich liposomes are induced via the classical and / or lectin pathways. This can be concluded from the complete inhibition of lysis of these liposomes in the presence of EGTA. If the alternative pathway were involved, some lysis would remain. The question of whether this type of liposome is specific for one complement pathway, and whether it is the classical or lectin pathway, is answered in Figures 11-21, which show that cholesterol-rich liposomes are specifically triggered via the classical pathway. [Figure 5] This figure shows a time-resolved complement assay investigating anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of 5G9 <anti-C3b> antibody. The antibody was incubated with serum (30 min on ice) before addition to the wells. The final "in-well" antibody concentrations are shown. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. As shown, antibody 5G9 blocks the complement system very effectively. The literature states that it should inhibit the classical pathway. Based on the literature we found, it is unclear whether it also inhibits the lectin pathway. This set of data indicates that antibodies against specific complement proteins function effectively as complement inhibitors. [Figure 6]Normalized endpoint data (60 min) from a time-resolved complement assay examining anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of 5G9 antibody are shown. The antibody was incubated with serum (30 min on ice) before addition to the wells. Final "in-well" antibody concentrations are shown. Normalized to a positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. As shown, antibody 5G9 blocks the complement system very effectively. The literature states that it should inhibit the classical pathway. Based on the literature found, it is unclear whether it also inhibits the lectin pathway. This body of data indicates that antibodies against specific complement proteins function effectively as complement inhibitors. [Figure 7]Complement assay. A), B) Normalized endpoint data (60 min) of active serum samples, and C) lysis inhibition values ​​from a time-resolved complement assay investigating anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of 5G9 antibody. Antibody was incubated with serum (30 min on ice) before addition to the wells. Final "in-well" antibody concentrations are shown. In A) and B), normalization was performed to the positive control (30 mM OG + aS) after 60 min. In C), lysis inhibition was calculated by normalizing the endpoint value (60 min) of the active serum sample to the positive control without inhibitor. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. As shown, antibody 5G9 blocks the complement system very effectively. The literature suggests that it should inhibit the classical pathway. Based on the literature we found, it is unclear whether it also inhibits the lectin pathway. This body of data indicates that antibodies against specific complement proteins can function effectively as complement inhibitors. [Figure 8]Time-resolved complement assays are shown for anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1s> M81 antibody. Additionally, 1 μg / mL and 4 μg / mL 5G9 <anti-C3b> antibody samples are added as controls. The antibody is incubated with serum (on ice for 30 minutes) before addition to the wells. The final "in-well" antibody concentrations are shown. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. As shown, the <anti-C1s> antibody M81 does not affect complement activity. Although it has been reported in the literature that this antibody binds to C1s and thus blocks C4 cleavage and the classical pathway, this cannot be confirmed by our liposome assays, etc. However, it should be noted that we were unable to find any functional assays detailing the inhibitory effect of this antibody using the erythrocyte assay. [Figure 9] Normalized endpoint data (60 min) from a time-resolved complement assay investigating anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1s> M81 antibody are shown. Additionally, 1 μg / mL and 4 μg / mL 5G9 <anti-C3b> antibody samples were added as controls. The antibody was incubated with serum (on ice for 30 min) before addition to the wells. Final "in-well" antibody concentrations are shown. Normalized to a positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 10]Normalized endpoint data (60 min) of active serum samples from a time-resolved complement assay examining anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of anti-C1s M81 antibody are shown. Antibody was incubated with serum (30 min on ice) before addition to the wells. Final "in-well" antibody concentrations are shown. Normalized to a positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 11] Time-resolved complement assays are shown for anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1q85> antibody. Additionally, 1 μg / mL and 4 μg / mL 5G9 <anti-C3b> antibody samples are added as controls. The antibody is incubated with serum (on ice for 30 minutes) before addition to the wells. The final "in-well" antibody concentrations are shown. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 12] Normalized endpoint data (60 min) from a time-resolved complement assay using anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1q85> antibody are shown. Additionally, 1 μg / mL and 4 μg / mL 5G9 <anti-C3b> antibody samples were added as controls. The antibody was incubated with serum (on ice for 30 min) before addition to the wells. Final "in-well" antibody concentrations are shown. Normalized to a positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 13] Normalized endpoint data (60 min) of active serum samples from a time-resolved complement assay examining anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1q85> antibody are shown. Antibody was incubated with serum (30 min on ice) before addition to the wells. Final "in-well" antibody concentrations are shown. Normalized to a positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was IR35577. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 14] Time-resolved complement assays were performed on anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1q85> antibody. Additionally, a 4 μg / mL 5G9 <anti-C3b> antibody sample was added as a control. C1q-depleted serum was also measured. The antibody was incubated with serum (on ice for 30 minutes) before addition to the wells. The final "in-well" antibody concentrations are shown. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was pooled serum 1-4 or C1q-depleted serum (A300, CompTech). λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 15] Time-resolved complement assays were performed using anionic cholesterol-rich liposomes (42%, KH210127) with C1q-depleted serum. To ensure parity with antibody-containing samples, serum was incubated on ice for 30 min before addition to the wells. Pooled serum 1–4 was used to preserve C1q-depleted serum, and inactive serum and positive controls were prepared. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was pooled serum 1–4 or C1q-depleted serum (A300, CompTech). λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 16] Normalized endpoint data (60 min) from a time-resolved complement assay using anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1q85> antibody are shown. Additionally, a 4 μg / mL 5G9 <anti-C3b> antibody sample was added as a control. C1q-depleted serum was also measured. The antibody was incubated with serum (on ice for 30 min) before addition to the wells. The final "in-well" antibody concentration is shown. Normalized to the positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was pooled serum 1-4 or C1q-depleted serum (A300, CompTech). λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 17] Normalized endpoint data (60 min) from a time-resolved complement assay using C1q-depleted serum on anionic cholesterol-rich liposomes (42%, KH210127) are shown. Normalized to the positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. Serum sources were pooled serum 1-4 or C1q-depleted serum (A300, CompTech). λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 18] Normalized endpoint data (60 min) of active serum samples from a time-resolved complement assay examining anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1q85> antibody are shown. Antibody was incubated with serum (30 min on ice) before addition to the wells. Final "in-well" antibody concentrations are shown. Normalized to a positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was pooled serum 1–4. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 19] Lysis inhibition values ​​for a time-resolved complement assay performed on anionic cholesterol-rich liposomes (42%, KH210127) with varying concentrations of <anti-C1q85> antibody are shown. The antibody was incubated with serum (30 min on ice) before addition to the wells. The final "in-well" antibody concentration is shown. Lysis inhibition was calculated by subtracting the intensity of an inactive serum sample as background and normalizing the endpoint value (60 min) of the active serum sample to the positive control without inhibitor. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was pooled serum 1–4. λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 20] Time-resolved complement assays were performed using C1q-depleted serum with anionic cholesterol-rich (42%, KH210127, 1 μL tL), LPS (1% LPS, AG201006-1, 1 μL tL), and antibody-modified liposomes (AG210412-2, 0.5 mL %<anti-biotin>AB, 1 μL tL). As a control, all liposomes were also assayed with IR35577 serum. The antibody was incubated with the liposomes for 1 hour at room temperature. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was either IR35577 or C1q-depleted serum (A300, CompTech). λEx = 565(8) nm and λEm = 585(8) nm, 37°C, gain = 150, n = 3. [Figure 21]Normalized endpoint data (60 min) from a time-resolved complement assay using C1q-depleted serum for anionic cholesterol-rich (42%, KH210127), LPS (1% LPS, AG201006-1), and antibody-modified liposomes (AG210412-2, 0.5 mL % <anti-biotin> AB) are shown. As a control, all liposomes were also assayed using IR35577 serum. Antibodies were incubated with liposomes for 1 h at RT. Normalization was performed against a positive control (30 mM OG + aS) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 2% by volume per well. The serum source was either IR35577 or C1q-depleted serum (A300, CompTech). λEx=565(8) nm and λEm=585(8) nm, 37°C, gain=150, n=3. [Figure 22] This figure shows a time-resolved complement assay performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) using a dilution curve of C1-INH incubated with serum (60 min on ice) before addition to the wells. C1-INH was added to inhibit the classical and lectin pathways. Complement assays were performed in liposomal complement buffer. The serum content of all liposomes was 5% by volume per well. The C1-INH content reported is the concentration in the well. The serum source was Innovative Research human complement serum. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. As shown, lysis of LPS-modified liposomes is partially inhibited in the presence of C1-INH. C1-INH is known to inhibit both the classical and lectin pathways. Because LPS is used as the inducer, the alternative pathway is also involved, and therefore, some lysis remains (alternative pathway-associated lysis). This data set indicates that C1-INH is suitable as an inhibitor in liposomal complement assays and can be used in combination with LPS-modified liposomes for alternative pathway-specific lysis. [Figure 23]Time-resolved complement assay activity serum samples are shown for anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) containing C1-INH, which were incubated with serum (60 min on ice) before addition to the wells. C1-INH was added to inhibit the classical and lectin pathways. Complement assays were performed in liposomal complement buffer. The serum content of all liposomes was 5% by volume per well. The serum source was Innovative Research human complement serum. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 24] Figure 1 shows the fluorescence intensity values ​​after 60 minutes of complement assay-active serum samples tested on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) containing C1-INH, which were incubated with serum (60 minutes on ice) before addition to the wells. Complement assays were performed in liposomal complement buffer. The serum content of all liposomes was 5% by volume per well. The serum source was Innovative Research human complement serum. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 25] Normalized endpoint data (60 min) from a time-resolved complement assay is shown for anionic 1 mol% LPS liposomes (AG201006-1, 10 μM) containing C1-INH, incubated with serum (60 min on ice) before addition to the wells. Normalized to a positive control (30 mM OG + 10% aS by volume) after 60 min. Complement assays were performed in liposomal complement buffer. The serum content of all liposomes was 5% by volume per well. The serum source was Innovative Research human complement serum. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 26]This figure shows a time-resolved complement assay performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) preincubated with EGTA. EGTA was added at a 1:1 ratio with serum before well addition (5 min, RT) and finally diluted to 0–1 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 27] Normalized endpoint data (60 min) from a time-resolved complement assay performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) preincubated with EGTA are shown. EGTA was added at a 1:1 ratio with serum prior to well addition (5 min, RT) and final dilutions were made to 0–1 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Normalized to a positive control (30 mM OG + 10% aS, by volume) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 28] Active serum samples from a time-resolved complement assay were tested using anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) preincubated with EGTA. EGTA was added at a 1:1 ratio to serum before well addition (5 min, RT), and the final dilution was 0–1 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 29]Time-resolved complement assays were performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) preincubated with EGTA at varying MgCl2 levels (0.2–1 mM). EGTA was added at a 1:1 ratio with serum prior to well addition (5 min, RT) and final dilution to 0 or 0.4 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 30] Normalized endpoint data (60 min) from a time-resolved complement assay was performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) pre-incubated with EGTA at varying MgCl2 levels (0.2–1 mM). EGTA was added at a 1:1 ratio with serum prior to well addition (5 min, RT) and final dilution to 0 or 0.4 mM EGTA. Normalized to a positive control (30 mM OG + 10% aS, by volume) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 31] Active serum samples from a time-resolved complement assay were tested on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) pre-incubated with EGTA at varying MgCl2 levels (0.2–1 mM). EGTA was added at a 1:1 ratio to serum prior to well addition (5 min, RT) and final dilution to 0 or 0.4 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 32] This figure shows a time-resolved complement assay performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) preincubated with EGTA. EGTA was added at a 1:1 ratio with serum prior to well addition (5 min, RT) and finally diluted to 0-0.4 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1-4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 33] Normalized endpoint data (60 min) from a time-resolved complement assay performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) preincubated with EGTA are shown. EGTA was added at a 1:1 ratio with serum prior to well addition (5 min, RT) and diluted to a final concentration of 0–0.4 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Data were normalized to a positive control (30 mM OG + 10% aS, by volume) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 34] Active serum samples from a time-resolved complement assay were tested using anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) preincubated with EGTA. EGTA was added at a 1:1 ratio to serum before well addition (5 min, RT), and the final dilution was 0–0.4 mM EGTA. EGTA was added to inhibit the classical and lectin pathways. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 35]Time-resolved complement assays were performed on anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) with varying EGTA inactivation times (0–35 min). EGTA was incubated 1:1 with serum prior to addition to the wells. To inhibit the classical and lectin pathways, the concentration was kept constant in each well (except the control) at 0.4 mM EGTA in LCB containing 1 mM MgCl2. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 36] Active serum samples from a time-resolved complement assay were tested with anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) at varying EGTA inactivation times (0–35 min). EGTA was incubated 1:1 with serum prior to addition to the wells. To inhibit the classical and lectin pathways, the concentration was constant in each well (except the control) at 0.4 mM EGTA in LCB containing 1 mM MgCl2. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. [Figure 37]Normalized endpoint data (60 min) from a time-resolved complement assay using anionic 1 mol% LPS liposomes (AG201006-1, 10 μL) with varying EGTA inactivation times (0–35 min) are shown. EGTA was incubated 1:1 with serum prior to addition to the wells, and the concentration was constant in each well (except the control) at 0.4 mM EGTA in LCB containing 1 mM MgCl2 to inhibit classical and lectin pathways. Normalized data were obtained against a positive control (30 mM OG + 10% aS by volume) after 60 min. Complement assays were performed in liposome complement buffer. The serum content of all liposomes was 10% by volume per well. The serum source was pooled serum 1–4. λEx = 565 nm and λEm = 585 nm, 37°C, gain = 150, n = 3. DETAILED DESCRIPTION OF THE INVENTION

[0045] It is an object of the present invention to provide efficient complement assays, such as efficient methods for determining complement activity. For example, it is an object of the present invention to provide standardized, inexpensive, rapid, and high-throughput assays for the complement pathways. It is a further object of the present invention to provide means for specifically determining the activity of each of the three complement pathways.

[0046] The present invention relates to methods for determining complement activity, comprising A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity. In one embodiment, the method for determining complement activity comprises determining total complement activity, particularly determining classical complement pathway activity, alternative complement pathway activity, and lectin complement pathway activity. In one embodiment, total complement activity includes or consists of complement activity of all three complement pathways, particularly the classical complement pathway, the alternative complement pathway, and the lectin complement pathway. In one embodiment, the method for determining complement activity is an in vitro and / or ex vivo method for determining complement activity.

[0047] In one embodiment, determining the classical complement pathway activity comprises providing one or more liposomes comprising a marker; optionally, a liposome for determining classical complement pathway activity as defined herein, particularly a liposome for determining classical complement pathway activity of a collection of liposomes as defined herein.

[0048] In one embodiment, the one or more liposomes provided in step A)a), particularly the one or more liposomes for assessing classical complement pathway activity, contain cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol, even more preferably about 40 mol% to about 60 mol%, for example, about 40 mol% to about 50 mol% or about 40 mol% to about 45 mol% cholesterol. The present inventors have found that liposomes containing at least 30 mol% cholesterol, particularly liposomes containing 40 mol% or more cholesterol, advantageously activate the classical complement pathway. Therefore, liposomes containing at least 30 mol% cholesterol, particularly liposomes containing 40 mol% or more cholesterol, can advantageously be used to assess classical complement pathway activity. Alternatively or additionally, to activate the classical complement pathway, the one or more liposomes provided in step A)a), particularly the one or more liposomes for assessing classical complement pathway activity, may contain a classical complement pathway activator. In one embodiment, the one or more liposomes provided in step A)a), particularly the one or more liposomes for determining classical complement pathway activity, comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin. In one embodiment, the one or more liposomes provided in step A)a) are configured to activate the classical complement pathway by comprising at least 30 mol% cholesterol, preferably at least 40 mol% cholesterol, and / or by comprising a classical complement pathway activator. In one embodiment, when the one or more liposomes provided in step A)a) comprise less than 30 mol% cholesterol, particularly less than 40 mol% cholesterol, the one or more liposomes comprise a classical complement pathway activator. Advantageously, the one or more liposomes provided in step A)a) can be configured to activate the classical complement pathway with an appropriate cholesterol content, preferably at least 40 mol% cholesterol and / or with a classical complement pathway activator.The present inventors have found that liposomes containing at least 30 mol % cholesterol, particularly 40 mol % or more cholesterol, and / or containing a classical complement pathway activator advantageously enable specific determination of classical complement activity.

[0049] In one embodiment, the liposomes for determining classical complement pathway activity of the liposome collection of the present invention are liposomes as defined in step A)a) of the method for determining complement activity of the present invention. In one embodiment, the one or more liposomes provided in step A)a) comprise cholesterol, e.g., at least 30 mol% cholesterol, and a phospholipid; preferably, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol). In one embodiment, the one or more liposomes provided in step A)a) comprise at least 30 mol% cholesterol, preferably at least 40 mol% cholesterol, 10 mol% to 30 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 25 mol% to 45 mol% 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol). In one embodiment, the one or more liposomes provided in step A)a) comprise 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine.

[0050] In one embodiment, determining the alternative complement pathway activity comprises providing one or more liposomes comprising a marker; optionally, a liposome for determining alternative complement pathway activity as defined herein, particularly a liposome for determining alternative complement pathway activity of a collection of liposomes defined herein.

[0051] In one embodiment, the one or more liposomes provided in step B)a), particularly the one or more liposomes for determining alternative complement pathway activity, comprise an alternative complement pathway activator, preferably lipopolysaccharide, more preferably about 0.01 mol% to about 5 mol% lipopolysaccharide, even more preferably about 1 mol% lipopolysaccharide. Advantageously, liposomes containing an alternative complement pathway activator, particularly in combination with a classical complement pathway inhibitor and a lectin complement pathway inhibitor, enable specific determination of alternative complement pathway activity. In one embodiment, the one or more liposomes provided in step B)a), particularly the one or more liposomes for determining alternative complement pathway activity, comprise cholesterol, preferably ≦10 mol% cholesterol. The inventors have found that cholesterol can provide advantageous stability of the liposomes. In particular, the inventors have found that liposomes containing ≦10 mol% cholesterol are advantageously stable and do not specifically activate the classical complement pathway. In one embodiment, the lipopolysaccharide is present in the liposome in a range of about 0.001% to about 10% by weight, preferably about 0.01% to about 2% by weight, and more preferably about 1% by weight.

[0052] In one embodiment, the liposomes for determining alternative complement pathway activity of the liposome collection of the invention are liposomes as defined in step B)a) of the method for determining complement activity of the invention. In one embodiment, the one or more liposomes provided in step B)a) comprise phospholipids and cholesterol, preferably ≦10 mol% cholesterol; preferably 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol. In one embodiment, the one or more liposomes provided in step B)a) comprise 65 mol% to 85 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 10 mol% to 30 mol% 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), and ≦10 mol% cholesterol. In one embodiment, the one or more liposomes provided in step B)a) comprise a classical complement pathway inhibitor, preferably an anti-C1q85 antibody or antigen-binding fragment thereof. In one embodiment, the one or more liposomes provided in step B)a) comprise 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine.

[0053] In one embodiment, determining the lectin complement pathway activity comprises providing one or more liposomes comprising a marker; optionally, a liposome for determining lectin complement pathway activity as defined herein, particularly a liposome for determining lectin complement pathway activity of a liposome collection defined herein.

[0054] In one embodiment, the one or more liposomes provided in step C)a), particularly the one or more liposomes for determining lectin complement pathway activity, comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid. In one embodiment, the lectin complement pathway activator comprises or consists of a sugar moiety, an acetyl moiety, and / or a sialic acid.

[0055] Advantageously, liposomes containing a lectin complement pathway activator, particularly in combination with a classical and / or alternative complement pathway inhibitor, allow for specific determination of lectin complement pathway activity. In one embodiment, the one or more liposomes provided in step C)a), particularly the one or more liposomes for determining lectin complement pathway activity, comprise cholesterol, preferably ≦10 mol% cholesterol. The inventors have found that cholesterol can provide advantageous stability of the liposomes. In particular, the inventors have found that liposomes containing ≦10 mol% cholesterol are advantageously stable and do not specifically activate the classical complement pathway.

[0056] In one embodiment, the liposomes for determining lectin complement pathway activity of the liposome collection of the present invention are liposomes as defined in step C)a) of the method for determining complement activity of the present invention. In one embodiment, the one or more liposomes provided in step C)a) comprise phospholipids and ≦10 mol% cholesterol; preferably, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol. In one embodiment, the one or more liposomes provided in step C)a) comprise 65 mol% to 85 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 10 mol% to 30 mol% 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), and ≦10 mol% cholesterol. In one embodiment, the one or more liposomes provided in step C)a) comprise a classical complement pathway inhibitor, preferably an anti-C1q85 antibody or antigen-binding fragment thereof. In one embodiment, the one or more liposomes provided in step C)a) comprise 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine.

[0057] In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) are selected from anionic liposomes, PEGylated liposomes, and cationic liposomes, preferably anionic liposomes. In one embodiment, the cationic liposomes comprise 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine. In one embodiment, the one or more liposomes provided in step A)a), step B)a), and / or step C)a) comprise or consist of anionic liposomes. In one embodiment, the one or more liposomes provided in step A)a), step B)a), and / or step C)a) comprise or consist of cationic liposomes, preferably comprising 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine. For example, the cationic liposome may contain cholesterol; preferably in the range of 65 mol% to 85 mol%, e.g., about 76%, of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; and preferably in the range of 10 mol% to 30 mol% of 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine. In one embodiment, the PEGylated liposome is a cationic liposome, anionic liposome, neutral liposome, and / or zwitterionic liposome. In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) contain 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and / or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). In one embodiment, the one or more liposomes provided in step A)a), the one or more liposomes provided in step B)a), and / or the one or more liposomes provided in step C)a) comprise lipids having a functional group, preferably a functional group selected from an azide group, a maleimide group, and an SH group.

[0058] In one embodiment, the one or more liposomes provided in step A)a), step B)a), and / or step C)a) comprise a glutaryl moiety, which can advantageously be used to covalently attach additional components, such as ligands, to the liposome.

[0059] In one embodiment, the one or more liposomes provided in step A)a), step B)a), and / or step C)a) of the method of the invention and / or liposome(s) A), B), and / or C) of the liposome collection of the invention comprise a complement activator. The term "complement activator", as used herein (e.g., in the context of liposome(s) provided in the method of the invention or in the context of liposome(s) of the liposome collection), relates to an agent, e.g., a molecule, that activates the complement system, in particular an agent, e.g., a molecule, that activates at least one component and / or at least one pathway of the complement system.

[0060] In one embodiment, the one or more liposomes provided in step A)a) of the method of the invention and / or liposome A) of the liposome collection of the invention comprise a classical complement pathway activator. The term "classical complement pathway activator," as used herein (e.g., in the context of liposome(s) provided in the method of the invention or in the context of liposome(s) of the liposome collection), refers to an agent, e.g., a molecule, that activates the classical complement pathway, particularly an agent, e.g., a molecule, that activates at least one component of the classical complement pathway, optionally FITC and / or digoxigenin. In one embodiment, the classical complement pathway activator is selected from an antibody, an antigen, and a ligand such as biotin or streptavidin.

[0061] In one embodiment, the one or more liposomes provided in step B)a) of the method of the invention and / or liposome B) of the liposome collection of the invention comprise an alternative complement pathway activator. The term "alternative complement pathway activator," as used herein (e.g., in the context of liposome(s) provided in the method of the invention or in the context of liposome(s) of the liposome collection), relates to an agent, e.g., a molecule, that activates the alternative complement pathway, particularly an agent, e.g., a molecule, that activates at least one component of the alternative complement pathway. In one embodiment, the alternative complement pathway activator is selected from lipopolysaccharides.

[0062] In one embodiment, the one or more liposomes provided in step C)a) of the method of the invention and / or liposome C) of the liposome collection of the invention comprise a lectin complement pathway activator. The term "lectin complement pathway activator," as used herein (e.g., in the context of liposome(s) provided in the method of the invention or in the context of liposome(s) of the liposome collection), relates to an agent, e.g., a molecule, that activates the lectin complement pathway, particularly an agent, e.g., a molecule, that activates at least one component of the lectin complement pathway. In one embodiment, the lectin complement pathway activator is selected from a sugar moiety, an acetyl moiety, and / or sialic acid.

[0063] In one embodiment, determining the classical complement pathway activity comprises contacting the one or more liposomes and / or the sample with a classical complement pathway activator, e.g., a classical complement pathway activator selected from an antibody, such as an anti-biotin antibody, an antigen, and a ligand, such as biotin or streptavidin. In one embodiment, determining the alternative complement pathway activity comprises contacting the one or more liposomes and / or the sample with an alternative complement pathway activator. In one embodiment, determining the lectin complement pathway activity comprises contacting the one or more liposomes and / or the sample with a lectin complement pathway activator. For example, the activator may be added to a solution, such as a buffer, containing the sample and / or the liposomes.

[0064] In one embodiment, determining the classical complement pathway activity comprises contacting the sample and / or the one or more liposomes with an alternative complement pathway inhibitor and / or a lectin complement pathway inhibitor. In one embodiment, determining the alternative complement pathway activity comprises contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or a lectin complement pathway inhibitor. In one embodiment, determining the lectin complement pathway activity comprises contacting the sample and / or the one or more liposomes with an alternative complement pathway inhibitor and / or a classical complement pathway inhibitor. For example, the inhibitor may be added to a solution, such as a buffer, containing the sample and / or the liposomes.

[0065] The term "sample", as used herein, relates to a sample to be tested, preferably a patient sample. In one embodiment, the sample is selected from a blood sample such as a serum sample, a urine sample, a tear(s) sample, an aqueous humor sample, a nasal swab sample, a synovial fluid sample, a pleural effusion sample, an ascites sample, a cerebrospinal fluid sample, an exudate sample, an interstitial fluid sample, a cell culture supernatant, and any combination thereof; preferably, the sample is a serum sample. In one embodiment, the sample is a sample obtained from a patient and / or a patient sample, preferably a serum sample. In one embodiment, the patient sample is a serum sample, optionally a diluted serum sample. In one embodiment, the sample is a sample containing or suspected of containing one or more active components of the complement system, such as one or more active components of the classical complement pathway, one or more active components of the alternative complement pathway, and / or one or more active components of the lectin complement pathway. In one embodiment, the sample is a human serum sample. In one embodiment, the sample is not a guinea pig serum sample.

[0066] In one embodiment, contacting the one or more liposomes with the sample in steps A)b), B)c), and / or C)c) comprises incubating the one or more liposomes with the sample. In one embodiment, the contacting, particularly the incubation, is carried out at a temperature ranging from about 4°C to about 50°C, preferably from about 20°C to about 40°C, more preferably from about 30°C to about 39°C, for example, at about 37°C. In one embodiment, the contacting, particularly the incubation, is carried out for about 1 second to about 120 minutes, preferably from about 5 minutes to about 90 minutes, more preferably from about 15 minutes to about 60 minutes.

[0067] In one embodiment, the contacting is configured to cause the one or more active complement system components, if present in the sample, to lyse the one or more liposomes. In one embodiment, an "active" complement system component is a fully functional complement system component, preferably having the same functionality as and / or the physiological functionality of the respective complement system components in a healthy individual. In one embodiment, the terms "active complement system component," "fully functional complement system component," and "physiological complement system component" are used interchangeably. In one embodiment, lysis of the one or more liposomes, particularly lysis of the liposomes induced by active complement system components, releases the marker from the one or more liposomes. In one embodiment, the term "active" in the context of the complement system or complement pathway refers to functional complement, e.g., physiological complement.

[0068] In one embodiment, detecting the released marker in steps A)c), B)d), and / or C)d) includes any of fluorescent, colorimetric, chemiluminescent, electrochemiluminescent, electrochemical, bioluminescent, and / or visual detection of the marker, optionally including detection using a mobile phone. In one embodiment, the term "detecting the released marker" as used herein includes direct and / or indirect detection of the released marker, particularly direct and / or indirect detection of the marker signal using, for example, fluorescent, colorimetric, chemiluminescent, electrochemiluminescent, electrochemical, bioluminescent, and / or visual detection, optionally including detection using a mobile phone. For example, the marker signal can be detected via i) direct detection, for example, of a fluorescent or bioluminescent signal; ii) pre-immobilization on the surface of liposomes followed by subsequent detection; and / or iii) pre-recovery of the liposomes from solution followed by subsequent detection. An advantage of the method of the present invention is that it can be performed outside a central laboratory, such as a doctor's office, without the need for expensive equipment. In one embodiment, the methods of the invention are performed as on-site and / or point-of-care assays.

[0069] In one embodiment, detecting the released marker in steps A)c), B)d), and / or C)d) comprises detecting the absence or presence of a released marker, preferably wherein the absence of a released marker indicates complement inactivity and the presence of a released marker indicates complement activity. In one embodiment, the method of determining complement activity comprises determining complement activity if the presence of a released marker is detected. In one embodiment, the method of determining complement activity comprises determining complement inactivity if no released marker and / or the absence of a released marker is detected.

[0070] In one embodiment, classical complement pathway activity is determined if the presence of the released marker is detected in step A)c), and / or classical complement pathway inactivity is determined if the absence of the released marker is detected in step A)c). In one embodiment, alternative complement pathway activity is determined if the presence of the released marker is detected in step B)d), and / or alternative complement pathway inactivity is determined if the absence of the released marker is detected in step B)d). In one embodiment, lectin complement pathway activity is determined if the presence of the released marker is detected in step C)d), and / or lectin complement pathway inactivity is determined if the absence of the released marker is detected in step C)d).

[0071] In one embodiment, detecting the released marker in steps A)c), B)d), and / or C)d) comprises directly or indirectly detecting the released marker. In one embodiment, indirectly detecting the released marker comprises providing the one or more liposomes comprising a marker, e.g., a chemiluminescent marker, in the form of immobilized liposome(s) and detecting the marker contained in the immobilized liposome(s) after removing the released marker, e.g., by a washing step. In one embodiment, the liposomes are immobilized, e.g., via biotin on the liposome and streptavidin on the surface, or via digoxigenin on the liposome and anti-digoxigenin antibody on the surface, or via FITC on the liposome and anti-FITC immobilized on the surface. In one embodiment, the method comprises immobilizing the one or more liposomes provided in steps A)a), B)a), and / or C)a). In one embodiment, detecting the released marker in steps A)c), B)d), and / or C)d) comprises detecting the absence or presence of the released marker. In one embodiment, detecting the released marker in steps A)c), B)d), and / or C)d) comprises detecting the absence of the released marker, the absence of which preferably indicates the presence of non-lysed liposomes, and / or detecting the presence of the released marker, the presence of which preferably indicates the presence of lysed liposomes. In one embodiment, the method comprises separating non-lysed liposomes, such as immobilized non-lysed liposomes, from lysed liposomes. In one embodiment, the method of the present invention comprises separating lysed and non-lysed liposomes, preferably by lateral flow assay, filtration, chromatography, and / or centrifugation. In one embodiment, the method comprises detecting the released marker and detecting a marker contained in the non-lysed liposomes.In one embodiment, detecting the released marker in steps A)c), B)d), and / or C)d) preferably comprises detecting intact liposomes, in particular non-lysed liposomes, by detecting a marker contained in the intact liposomes. In one embodiment, the method comprises detecting a marker contained in the non-lysed liposome(s). In one embodiment, the method comprises detecting a marker present in the non-lysed liposome(s), such as an electrochemical marker, a colorimetric marker, a chemiluminescent marker, and / or an electrochemiluminescent marker.

[0072] In one embodiment, A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity comprises detecting a marker contained in non-lysed liposomes. For example, in the absence of complement activity, contacting the one or more liposomes with the sample in step A)b), step B)c), and / or step C)c) does not result in lysis of the one or more liposomes, i.e., the liposomes remain non-lysed. In one embodiment, the one or more liposomes provided in step A)a), step B)a), and / or step C)a) are non-lysed liposomes. In one embodiment, a marker contained in the non-lysed liposomes is detected. The inventors have found that, advantageously, the specific interaction of classical complement with the non-lysed liposomes provided in step A)a), the specific interaction of alternative complement with the non-lysed liposomes provided in step B)a), and / or the specific interaction of lectin complement with the non-lysed liposomes provided in step C)a) results in lysis of the respective liposomes and release of the marker. In one embodiment, the one or more liposomes contacted with the sample in step A)b), step B)c), and / or step C)c) remain non-lysed liposomes in the absence of complement activity.

[0073] In one embodiment, the step of detecting a marker contained in non-lysed liposomes comprises direct detection, e.g., by measuring the signal of the marker contained in the non-lysed liposomes, and / or comprises indirect detection, e.g., by first removing the lysed liposomes and / or released marker, then lysing the non-lysed liposomes and detecting the released marker in the liposomes. In one embodiment, the A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity preferably comprises a washing step of removing the lysed liposomes and / or released marker by washing, prior to the step of detecting the marker contained in the non-lysed liposomes. In one embodiment, detecting the released marker in steps A)c), B)d), and / or C)d) comprises detecting the released marker, e.g., by direct detection of the marker contained in the non-lysed liposomes. In one embodiment, the A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity comprises detecting a marker contained in non-lysed liposomes, wherein prior to detecting the marker contained in the non-lysed liposomes, the lysed liposomes and / or released marker are removed, e.g., washed away, and then the non-lysed liposomes are lysed to release the marker and the released marker is detected.

[0074] In one embodiment, determining the alternative complement pathway activity comprises contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor. In one embodiment, the classical complement pathway inhibitor and the lectin complement pathway inhibitor are the same inhibitor or different inhibitors. In one embodiment, the classical complement pathway inhibitor and the lectin complement pathway inhibitor comprise or consist of the same compound or different compounds. For example, a compound such as EGTA may inhibit both the classical complement pathway and the lectin complement pathway. In one embodiment, the inhibitor that inhibits the classical complement pathway and the lectin complement pathway is selected from EGTA, C1 inhibitor, and C4BP. In one embodiment, the inhibitor that is a classical complement pathway inhibitor and a lectin complement pathway inhibitor is selected from EGTA, C1 inhibitor, and C4BP. In one embodiment, classical complement pathway inhibitors, particularly classical complement pathway inhibitors used in assessing alternative complement pathway activity, include EGTA, an anti-C1q85 antibody or an antigen-binding fragment thereof, C1 inhibitor, and / or C4BP, preferably EGTA and / or C1 inhibitor. In one embodiment, a specific classical complement pathway inhibitor, particularly a classical complement pathway inhibitor that inhibits only the classical complement pathway, is an anti-C1q85 antibody or an antigen-binding fragment thereof. In one embodiment, a lectin complement pathway inhibitor includes EGTA, C1 inhibitor, an anti-MASP antibody, and / or C4BP, preferably EGTA and / or C1 inhibitor. In one embodiment, a specific lectin complement pathway inhibitor, particularly a lectin complement pathway inhibitor that inhibits only the lectin complement pathway, is an anti-MASP antibody.

[0075] In a preferred embodiment, the classical complement pathway inhibitor and the lectin complement pathway inhibitor comprise or consist of EGTA. The advantage of EGTA is that it efficiently inhibits both the classical complement pathway and the lectin complement pathway. Furthermore, EGTA inhibits both the classical complement pathway and the lectin complement pathway at low cost. Advantageously, when the classical complement pathway and the lectin pathway are inhibited by the classical complement pathway inhibitor and the lectin complement pathway inhibitor, the activity of the alternative complement pathway can be specifically determined. The alternative pathway specificity of the method for determining complement activity of the present invention is greatly enhanced when the classical pathway and the lectin pathway are inhibited by the classical complement pathway inhibitor and the lectin complement pathway inhibitor, such as EGTA.

[0076] In one embodiment, the term "classical complement pathway inhibitor," as used herein (e.g., in the context of the liposome(s) provided in the methods of the invention or in the context of the liposome(s) of the liposome collection), relates to any inhibitor of the classical complement pathway, for example, an inhibitor selected from EGTA, an anti-C1q85 antibody or an antigen-binding fragment thereof, C1-inhibitor, C4BP, and combinations thereof. In one embodiment, particularly when used in the context of determining lectin complement pathway activity, the classical complement pathway inhibitor is a specific classical complement pathway inhibitor, preferably selected from an anti-C1q85 antibody or an antigen-binding fragment thereof. In one embodiment, the term "lectin complement pathway inhibitor," as used herein (e.g., in the context of the liposome(s) provided in the methods of the invention or in the context of the liposome(s) of the liposome collection), relates to any inhibitor of the lectin complement pathway, for example, an inhibitor selected from EGTA, C1-inhibitor, an anti-MASP antibody, C4BP, and combinations thereof. In one embodiment, the lectin complement pathway inhibitor is a specific lectin complement pathway inhibitor, particularly when used in the context of determining classical complement pathway activity. When referring to an antibody in the context of the present invention, e.g., when referring to an inhibitor or activator comprising or consisting of an antibody, it is understood that reference to such an antibody also relates to fragments thereof, particularly antigen-binding fragments thereof. In one embodiment, the term "alternative complement pathway inhibitor," as used herein (e.g., in the context of the liposome(s) provided in the methods of the present invention or in the context of the liposome(s) of the liposome collection), relates to any inhibitor of the alternative complement pathway, e.g., an inhibitor selected from complement factor H, mini-complement factor H, anti-factor B antibody or antigen-binding fragment thereof, anti-properdin antibody or antigen-binding fragment thereof, and any combination thereof. In one embodiment, each of the classical complement pathway inhibitor, the lectin complement pathway inhibitor, and the alternative complement pathway inhibitor is a specific inhibitor.In one embodiment, the anti-MASP antibody is a human monoclonal antibody that targets mannan-binding lectin-associated serine protease-2, such as narsoplimab. The term "C4BP" as used herein refers to C4b-binding protein.

[0077] In one embodiment, step B)b) comprises the step of: 2+ The sample and / or the one or more liposomes are treated with Ca in the presence of at least 0.001 mM MgCl, preferably in the presence of at least 0.1 mM MgCl, even more preferably in the presence of at least 0.5 mM MgCl, for example about 1 mM MgCl. 2+ In one embodiment, determining the alternative complement pathway activity comprises contacting the antibody with a complexing agent, preferably a classical complement pathway inhibitor and / or a lectin complement pathway inhibitor selected from EGTA. In one embodiment, the EGTA and the Mg have a molar ratio of about 1:1. In one embodiment, determining the alternative complement pathway activity comprises contacting the antibody with a complexing agent, preferably a classical complement pathway inhibitor and / or a lectin complement pathway inhibitor selected from EGTA and the Mg ... 2+ The method includes contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor, preferably EGTA, in the presence of at least 0.001 mM MgCl2, preferably at least 0.1 mM MgCl2, and even more preferably at least 0.5 mM MgCl2, e.g., about 1 mM MgCl2. When a classical complement pathway inhibitor and a lectin complement pathway inhibitor are used, liposome lysis occurs solely based on alternative complement pathway activity. In a preferred embodiment, EGTA is provided in an amount of at least 0.2 mM. In one embodiment, EGTA is provided in an amount of at least 0.2 mM, preferably at least 0.4 mM, preferably in combination with 1 mM MgCl2. In one embodiment, the classical complement pathway inhibitor and / or the lectin complement pathway inhibitor is EGTA at a concentration of at least 0.2 mM, preferably at least 0.4 mM. In one embodiment, the classical and / or lectin complement pathway inhibitor is EGTA at a concentration ranging from about 0.2 mM to about 1 mM, preferably from about 0.2 to about 0.8 mM, and optionally 1 mM Mg 2+In one embodiment, step B)b) comprises treating the sample and / or the one or more liposomes with Ca in an amount of preferably at least 0.2 mM, more preferably at least 0.4 mM. 2+ A complexing agent, optionally EGTA, and Mg 2+ , preferably in the presence of at least 0.001 mM MgCl2, preferably in the presence of at least 0.1 mM MgCl2, even more preferably in the presence of at least 0.5 mM MgCl2, for example about 1 mM MgCl2.

[0078] In one embodiment, determining the lectin complement pathway activity comprises contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor. In one embodiment, the classical complement pathway inhibitor and the alternative complement pathway inhibitor are the same inhibitor or different inhibitors. In one embodiment, the classical complement pathway inhibitor and the alternative complement pathway inhibitor comprise or consist of the same compound or different compounds. In one embodiment, the classical complement pathway inhibitor, particularly the classical complement pathway inhibitor used in determining lectin complement pathway activity, comprises or consists of an anti-C1q85 antibody or antigen-binding fragment thereof. In one embodiment, the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, anti-factor B antibody or antigen-binding fragment thereof, anti-properdin antibody or antigen-binding fragment thereof, and any combination thereof. Advantageously, inhibition of the classical complement pathway and / or the alternative complement pathway, respectively, with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor provides highly lectin pathway specificity.

[0079] Each of the determination of classical complement pathway activity, the determination of alternative complement pathway activity, and the determination of lectin complement pathway activity includes contacting the one or more liposomes with the sample and allowing the one or more active complement system components, if present in the sample, to lyse the one or more liposomes. In particular, when determining classical complement pathway activity, if one or more active complement system components of the classical complement pathway are present in the sample, the one or more liposomes, particularly the liposome(s) for determining classical complement pathway activity, are lysed. In particular, when determining alternative complement pathway activity, if one or more active complement system components of the alternative complement pathway are present in the sample, the one or more liposomes, particularly the liposome(s) for determining alternative complement pathway activity, are lysed. In particular, when determining lectin complement pathway activity, if one or more active complement system components of the lectin complement pathway are present in the sample, the one or more liposomes, particularly the liposome(s) for determining lectin complement pathway activity, are lysed. Dissolution of the liposome(s) releases a marker, which can be detected. For example, complement activity can be determined by analyzing how much of the marker is released. The higher the level of the released marker, the higher the complement activity. If complement is active, the liposomes are lysed. Thus, if complement is active, the released marker is detected. For example, if the classical complement pathway is active, the liposome(s) for determining classical complement pathway activity are lysed. For example, if the alternative complement pathway is active, the liposome(s) for determining alternative complement pathway activity are lysed. For example, if the lectin complement pathway is active, the liposome(s) for determining lectin complement pathway activity are lysed. In the absence of released marker and / or in the absence of liposome lysis, complement can be determined to be inactive.

[0080] In one embodiment, the A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity further comprises detecting a basal level of the marker. In one embodiment, the basal level of the marker is detected prior to the step of contacting the one or more liposomes with the sample, at a time point ranging from about 1 second to about 20 minutes, preferably about 1 second to about 15 minutes, after initial contact of the one or more liposomes with the sample, and / or using inactivated serum and / or buffer. For example, the basal level may include or be the marker level before complement activation, e.g., before the contact or before the activation, and / or may include or be the marker level without complement activity, e.g., the marker level of a negative control including inactivated serum and / or buffer. In one embodiment, detecting the basal level comprises performing a negative control. For example, determining the basal level may include determining the marker level in a buffer, particularly a serum-free buffer, determining the marker level in inactivated serum, and / or determining the marker level at a time point prior to complement activation. In one embodiment, the basal level is determined at a temperature ranging from about 4°C to about 50°C, preferably from about 20°C to about 40°C, more preferably from about 30°C to about 39°C, e.g., about 37°C; and / or for about 1 second to about 120 minutes, preferably from about 5 minutes to about 90 minutes, more preferably from about 15 minutes to about 60 minutes. Preferably, the inactivated serum is serum in which the complement system has been inactivated, for example, with EDTA, EGTA, heat, ultrasound, and / or a solvent such as a water-soluble organic solvent. In one embodiment, the serum is human serum. In one embodiment, the serum is not guinea pig serum.

[0081] In one embodiment, the method for determining competitive activity comprises determining complement activity by comparing the released marker detected in steps A)c), B)d), and C)d), respectively, with a basal level of the marker, preferably the basal level of the marker detected in the step of detecting the basal level of the marker, and / or with a negative control. In one embodiment, a higher level of the released marker detected in steps A)c), B)d), and C)d), respectively, compared to the basal level of the marker and / or the negative control, indicates complement activity. In one embodiment, the method for determining complement activity comprises determining complement activity if released marker is detected, particularly if released marker is detected in steps A)c), B)d), and / or C)d). In one embodiment, the method for determining complement activity comprises determining the absence of complement activity, particularly complement inactivation, if released marker is not detected, particularly if released marker is not detected in steps A)c), B)d), and / or C)d) and / or if the level of detected released marker corresponds to the level of released marker in the negative control.

[0082] In one embodiment, the released markers detected in steps A)c), B)d), and / or C)d) are compared to a reference signal and / or reference value. In one embodiment, the determining the complement activity, in particular determining classical complement pathway activity, determining alternative complement pathway activity, and / or determining lectin complement pathway activity, comprises comparing the released markers detected in steps A)c), B)d), and / or C)d) to a reference signal and / or reference value.

[0083] In one embodiment, the term "reference signal and / or reference value" as used herein relates to a signal and / or value obtained for a sample with normal complement activity, preferably a sample from a healthy individual. In one embodiment, the method for determining complement activity comprises determining complement activity if the released markers detected in steps A)c), B)d), and / or C)d), respectively, are equal to or greater than the reference signal and / or reference value. In one embodiment, the method for determining complement activity comprises determining the absence or decreased complement activity if the released markers detected in steps A)c), B)d), and / or C)d), respectively, are decreased compared to the reference signal and / or reference value.

[0084] In one embodiment, the method includes conducting a positive control and / or a negative control. In one embodiment, conducting a positive control includes contacting the one or more liposomes provided in steps A)a), B)a), and / or C)a) with a detergent and / or solvent, preferably a detergent, more preferably a detergent selected from n-octyl-β-d-glucoside, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (80) sorbitan monooleate. In one embodiment, the terms "2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol" and "Triton X-100" are used interchangeably. In one embodiment, the terms "polyoxyethylene (20) sorbitan monolaurate" and "Tween 20" are used interchangeably. In one embodiment, the terms "polyoxyethylene (80) sorbitan monooleate," "polyoxyethylene (20) sorbitan monooleate," and "Tween 80" are used interchangeably. In one embodiment, performing a negative control comprises replacing the sample, optionally the serum sample, in steps A)a), B)a), and / or C)a) with a negative control sample comprising or consisting of inactive serum.

[0085] In one embodiment, the marker is quenched as long as it is contained in the liposome. In one embodiment, if complement is inactive in the sample, the liposome does not dissolve, the marker is not released from the liposome, and the signal of the marker contained in the liposome remains quenched. In one embodiment, if complement is active in the sample, the liposome dissolves, and the marker is released, and the released marker is therefore detectable.

[0086] In one embodiment, the detecting in step A)c), step B)d), and / or step C)d) comprises or consists of time-resolved detection of the released markers. Time-resolved detection is highly advantageous in that it allows further details of the complement-related disorder to be assessed, such as the delay in complement activation. In one embodiment, the detecting in step A)c), step B)d), and / or step C)d) comprises or consists of time-resolved detection of the released markers, an increase in the released markers over time being indicative of complement activation.

[0087] The term "marker", as used herein, e.g., in the context of one or more liposome(s) provided in the methods of the invention or in the context of a liposome(s) of the liposome collection of the invention, relates to a molecule that can be detected, preferably quantitatively analyzed, via any detection and / or analytical method known to those skilled in the art, such as chromatography, electrophoresis, microscopy, photometry; spectroscopy such as atomic absorption spectroscopy, ultraviolet-visible spectroscopy, X-ray spectroscopy, fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy, nuclear magnetic resonance spectroscopy, photoelectron spectroscopy; mass spectroscopy, electrochemical analysis, calorimetry, sequencing, precipitation, or extraction, etc. In one embodiment, the marker is selected from the group consisting of fluorescent markers, preferably indocyanine green such as pyrenetetrasulfonic acid, sulforhodamine B, 2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, or carboxyfluorescein; electrochemical markers, preferably potassium hexaferricyanide, potassium hexaferrocyanide, or ruthenium hexamine; colorimetric markers, preferably sulforhodamine B; chemiluminescent markers, preferably luminol or a derivative thereof, such as mCOOH-luminol; electrochemiluminescent markers, preferably luminol or ruthenium bipyridyl; bioluminescent markers such as GFP, other bioluminescent proteins, or luciferase; and combinations thereof. In one embodiment, the marker is water-soluble. For example, the marker can be selected from a water-soluble fluorescent marker, a water-soluble electrochemical marker, a water-soluble chemiluminescent marker, and a water-soluble bioluminescent marker. In one embodiment, the marker is selected from a fluorescent marker, an electrochemical marker, and a combination thereof. In a preferred embodiment, the marker is selected from sulforhodamine B and mCOOH-luminol. In a preferred embodiment, the marker provides a signal only when released from the liposome.In another embodiment, the marker provides a signal only when contained inside the liposome, and the signal disappears when the marker is released from the liposome. In one embodiment, the term "luminol" as used herein relates to luminol and its derivatives, particularly its water-soluble derivatives such as mCOOH-luminol, preferably luminol and mCOOH-luminol, more preferably mCOOH-luminol. In one embodiment, the marker is not carboxyfluorescein.

[0088] In one embodiment, the one or more liposomes comprising a marker provided in step A)a) comprise a first marker, the one or more liposomes comprising a marker provided in step B)a) comprise a second marker, and the one or more liposomes comprising a marker provided in step C)a) comprise a third marker, wherein the first marker, the second marker, and the third marker are different markers. Advantageously, the use of the first marker, the second marker, and the third marker allows for specific and simultaneous determination of the activity of each of three different complement pathways.

[0089] In one embodiment, detecting the released markers in steps A)c), B)d), and / or C)d) is performed simultaneously or sequentially, preferably simultaneously, e.g., by determining the classical complement pathway activity, the alternative complement pathway activity, and the lectin complement pathway activity in different wells of a multi-well plate. For example, the simultaneous detection can be performed in separate containers, such as separate wells of a multi-well plate.

[0090] In one embodiment, the liposome collection and / or kit of the invention is / are used in a method of the invention. In one embodiment, a method of the invention is carried out using the liposome collection and / or kit of the invention.

[0091] The term "liposome collection," as used herein, refers to a collection, e.g., a group, of liposomes. In one embodiment, the terms "liposome collection" and "liposome assembly" can be used interchangeably. In one embodiment, the liposome collection includes one or more types of liposomes, particularly one or more types, preferably at least two types of liposomes, selected from liposomes for determining classical complement pathway activity, liposomes for determining alternative complement pathway activity, and liposomes for determining lectin complement pathway activity. For example, the liposome collection can include liposomes for determining classical complement pathway activity and liposomes for determining alternative complement pathway activity.

[0092] In one embodiment, the liposome collection of the present invention comprises: - A) liposomes for determining classical complement pathway activity as defined herein; - B) liposomes for determining alternative complement pathway activity as defined herein, and / or - C) Liposomes for determining lectin complement pathway activity as defined herein; Including, Optionally, including A) and B), A) and C), B) and C), or A), B) and C).

[0093] In one embodiment, the liposomes for determining classical complement pathway activity of the liposome collection are liposomes as defined in the context of step A)a) of the method of determining complement activity of the invention. In one embodiment, the liposomes for determining alternative complement pathway activity of the liposome collection are liposomes as defined in the context of step B)a) of the method of determining complement activity of the invention. In one embodiment, the liposomes for determining lectin complement pathway activity of the liposome collection are liposomes as defined in the context of step C)a) of the method of determining complement activity of the invention. It is meant to be understood that all embodiments defining the liposome(s) provided in steps A)a), B)a), and / or C)a) of the method of determining complement activity of the invention also apply to liposomes A), B), and C) of the liposome collection of the invention, respectively, and vice versa, for example, to embodiments defining a marker for the liposome(s) or a component of the liposome(s). For example, all embodiments defining the liposome(s) provided in step A)a) of the method of the present invention also apply to the liposome(s) A) of the liposome collection of the present invention, and vice versa. For example, all embodiments defining the liposome(s) provided in step B)a) of the method of the present invention also apply to the liposome(s) B) of the liposome collection of the present invention, and vice versa. For example, all embodiments defining the liposome(s) provided in step C)a) of the method of the present invention also apply to the liposome(s) C) of the liposome collection of the present invention, and vice versa.

[0094] In one embodiment, the liposome collection is used in a method for determining complement activity as defined herein. The present invention also relates to the use of the liposome collection as defined herein in a method for determining complement activity and / or a method for diagnosing, monitoring, or treating a complement-associated disorder.

[0095] In a further aspect, the present invention provides a method for producing a composition comprising: - a liposome collection as defined herein; - a buffer, preferably a liposomal complement buffer; - optionally a classical complement pathway inhibitor, an alternative complement pathway inhibitor, and / or a lectin complement pathway inhibitor; preferably EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP; more preferably EGTA and / or C1 inhibitor; - optionally inactivated serum; preferably serum inactivated by EDTA, EGTA, heat, ultrasound and / or solvents such as water-soluble organic solvents; optionally a surfactant, for example, a surfactant selected from n-octyl-β-d-glucoside, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polyoxyethylene(20) sorbitan monolaurate, and polyoxyethylene(80) sorbitan monooleate, preferably n-octyl-β-d-glucoside; optionally an osmotic agent, preferably a sucrose solution; - optionally instructions for determining complement activity, preferably instructions for determining classical complement pathway activity, alternative complement pathway activity, and / or lectin complement pathway activity; more preferably instructions for a method of determining complement activity as defined herein. The present invention relates to a kit for determining complement activity, comprising:

[0096] In one embodiment, the kit further comprises test strips and / or microtiter plates. In one embodiment, the kit is used in a method for determining complement activity as defined herein. The present invention also relates to the use of a kit as defined herein in a method for determining complement activity and / or in a method for diagnosing, monitoring, or treating a complement-associated disorder.

[0097] In one embodiment, the liposome complement buffer includes a HEPES buffer. In one embodiment, the liposome complement buffer does not include a gelatin veronal buffer (GVB). A liposome complement buffer including a HEPES buffer is less toxic than a liposome complement buffer including a gelatin veronal buffer (GVB) because GVB contains the toxic component barbital, which is not present in the HEPES buffer of the liposome complement buffer.

[0098] In one embodiment, the liposome complement buffer comprises 10 mM HEPES, 150 mM NaCl, 135 nM CaCl2, and 1 mM MgCl2, preferably at a pH of about 7.4. In one embodiment, the inactivated serum is serum in which the complement system has been inactivated and / or one or more, preferably all, components of the complement system have been inactivated. For example, inactivated serum can be provided by activating a serum sample with EDTA, such as 200 mM EDTA, and / or EGTA, such as 0.5 μL EGTA.

[0099] In one embodiment, the osmotic agent is a sugar, salt, or polymer that imparts a certain osmotic pressure to the liposome. In one embodiment, the osmotic agent is sucrose. Advantageously, the osmotic agent stabilizes the liposome. In one embodiment, the osmotic agent is configured to provide an osmotic pressure equal to or greater than the osmotic pressure of the aqueous core of the liposome, e.g., about 50-75 mmol / kg higher than the osmotic pressure of the aqueous core of the liposome. For example, the osmotic agent can be provided in a buffer used with the liposome.

[0100] In one embodiment, the liposome collection defined herein and / or kit defined herein for use in a method for diagnosing, monitoring, or treating a complement-related disorder, preferably a complement-related disease, is used to monitor a complement-related disorder. For example, the liposome collection and / or kit can be used to determine whether a pathological and / or inactive complement system will normalize to a functional, i.e., active, complement system when administered to a patient in need thereof with a complement therapeutic agent. By monitoring complement activity, for example, using the methods, kits, and / or liposome collections of the present invention for determining complement activity, treatment of a patient in need thereof can be tailored with respect to the amount of complement therapeutic agent required and / or the duration of treatment with the complement therapeutic agent required. Advantageously, the methods, kits, and / or liposome collections of the present invention for determining complement activity can be used in the context of personalized medicine, for example, by determining whether a patient is predicted to be successfully treated with a respective complement therapeutic agent. When used in a method for treating a complement-related disorder, the liposome collection and / or kit can be used to predict whether a complement therapeutic agent is likely to be effective in a patient. For example, the liposome collection and / or kit may be part of a treatment for a complement-associated disorder in a patient, in which case the liposome collection and / or kit is used to determine in vivo and / or ex vivo whether a complement therapeutic agent has an effect on the patient's complement prior to administering the complement therapeutic agent to the patient.

[0101] The present invention also relates to a liposome collection as defined herein and / or a kit as defined herein for use in a method for diagnosing a complement-associated disorder, preferably a complement-associated disease, comprising: i) providing a patient sample, preferably a serum sample; ii) carrying out a method for determining complement activity as defined herein using a liposome collection as defined herein or a kit as defined herein; iii) diagnosing a complement-associated disorder if the released marker is absent or if a low level of the released marker is detected compared to a positive control and / or compared to a baseline value; The present invention relates to a liposome collection and / or kit comprising:

[0102] Advantageously, the methods of the present invention, e.g., liposome assays, are homogeneous assays that do not require washing steps, such as heterogeneous assays. Upon incubation with a body sample, complement activity causes liposome lysis, which can then be detected. Detection can be achieved by a variety of different strategies, including optical and electrochemical assays.

[0103] In contrast to the known CH50 liposome assay, which can only detect the classical pathway, the method of the present invention has the advantage of being able to address the three complement pathways separately. This is possible because the liposomes are designed with a specific lipid composition, a specific encapsulating agent concentration, and / or specific ligands. For example, liposomes with a cholesterol content of >30 mol%, e.g., 44 mol%, specifically induce the classical pathway. Liposomes containing 5 mol% cholesterol and 1 mol% lipopolysaccharide can induce all three pathways, but the addition of C1 inhibitor or EGTA detects only the alternative pathway. Sugar moieties, Ac-BSA, or sialic acid structures can be used to determine the lectin pathway. Advantageously, the method of the present invention allows for specific targeting of one or more pathways of the complement system, e.g., the classical pathway or the alternative pathway.

[0104] In a preferred embodiment, the liposomes provided in step A)a) of the method of the present invention and / or the liposomes A) of the liposome collection of the present invention comprise at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol, in particular to specifically trigger the classical complement pathway. Advantageously, the classical pathway can be specifically targeted without any activator, i.e., simply by liposomes comprising a high amount of cholesterol, such as at least 30 mol% cholesterol, in particular at least 40 mol% cholesterol.

[0105] In a preferred embodiment, liposomes provided in step B)a) of the method of the present invention and / or liposomes B) of the liposome collection of the present invention contain up to 10 mol% cholesterol and further comprise an alternative complement pathway activator, in particular for specifically inducing the alternative complement pathway.In a preferred embodiment, liposomes provided in step C)a) of the method of the present invention and / or liposomes C) of the liposome collection of the present invention contain up to 10 mol% cholesterol and further comprise a lectin complement pathway activator, in particular for specifically inducing the lectin complement pathway.

[0106] A further advantage of the method of the present invention is that complement activity can be monitored in real time. For example, in the case of fluorescence detection, liposomes capture a high concentration of fluorescent molecules, which are self-quenching, so intact, i.e., undissolved, liposomes show minimal fluorescence signals. Upon dissolution, the dye is diluted into the surrounding solution, and a significant increase in fluorescence is observed. Therefore, while the liposomes are incubated with body fluids, the activity of the complement system can be easily monitored to obtain a real-time signal. The same can be done when capturing electrochemical markers.

[0107] A further advantage of the methods of the present invention is that they allow for multiplexed analysis of complement activity. For example, capturing different markers (e.g., three distinct fluorescent dyes or three different electrochemical markers) allows for simultaneous quantitative detection of each complement pathway separately. A further advantage of the methods of the present invention is that they are cost-effective methods for determining complement activity.

[0108] In one embodiment, the term "patient" relates to a human or animal, preferably a human. As used herein, the terms "of the invention," "in accordance with the invention," "inventive," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0109] As used herein, the term "comprising" is interpreted as encompassing both "including" and "consisting of," both of which are specifically contemplated according to the present invention and therefore individually disclosed embodiments. As used herein, "and / or" should be interpreted as specifically disclosing each of the two specified features or components, regardless of the presence or absence of the other feature or component. For example, "A and / or B" should be interpreted as specifically disclosing (i) A, (iii) B, and (iii) each of A and B, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, e.g., ±5% from the specified numerical value. As a person skilled in the art would understand, the specific deviation of the numerical value for a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have a larger deviation than artificial or engineered technical effects. Where an indefinite or definite article is used when referring to a singular noun e.g. "a", "an" or "the", the plural of that noun is also included unless something else is stated. [Example]

[0110] Reference will now be made to examples which are given to illustrate but not to limit the invention.

[0111] Example 1: Alternative Route The solution followed the following protocol: Liposomes (in this case, modified with 1 mol% lipopolysaccharide) ·buffer Liposomal Complement Buffer (LCB) 10mM HEPES, 150mM NaCl, 135nM CaCl2, 1mM MgCl2, pH7.4 Inactivation buffer (for negative control, to inactivate the complement system): Liposomal Inactivated Complement Buffer (iaCB) 90% liposomal complement buffer and 200 mM EDTA, 0.5 μL EGTA pH ~8 Inhibitor solution (to inactivate the classical and lectin pathways of the complement system) EGTA-containing liposome complement buffer, pH 7.4 or C1-Inh (for control experiments) Sucrose-containing liposome complement buffer (used in all wells to adjust the sucrose content in each well to ensure better stability of liposomes) Human serum Detergent-containing solution (used as a positive control to dissolve all liposomes) 300 mM n-octyl-β-d-glucoside (OG) in double-distilled water

[0112] Assay preparation: 1. Prepare liposome stock solution Mix the liposomes with buffer to obtain a 10x concentrated stock solution that will be needed later. 2. Prepare stock solutions of each inhibitor Mix inhibitor with buffer to obtain a 10x concentrated stock solution 3. Prepare a black microtiter plate on ice Add buffer to all wells (volumes will vary for buffer control, activated serum, negative control, and positive control, and all wells should be 100 μL when all solutions are added at the end) Add sucrose solution (1M stock, 20 μL per well) Add inactivation buffer (10 μL to each negative control well) 4. Add detergent solution (10 μL to each positive control well) 5. Preheat the fluorescence plate reader to 37°C. Steps for performing the assay: 6. Mix the serum with the respective amount of inhibitor (prepared in 2.) 7. Incubate the mixture for the respective time. C1-Inh: 60 minutes on ice (used only in control experiments) EGTA test: 5 minutes at room temperature (unless otherwise specified) 8. Meanwhile, add 10 μL of liposomes (prepared in 1.) to each well. 9. Add the appropriate amount of serum-inhibitor mixture to all samples containing serum (positive control, negative control, and activated serum; activated serum must be added last). The total volume in all wells must now be 100 μL. 10. Measuring time-resolved fluorescence intensity

[0113] Fluorescence intensity was measured using a BioTek SYNERGY neo2 fluorescence reader. The wavelength was λ Ex = 565(5) nm and λ Em = 585(5) nm. The measurement temperature in the instrument was constant at 37°C. Measurements were performed at a gain of 150. Measurements were performed for 60 minutes. For the first 15 minutes, measurements were taken at 1.5 minute intervals, with three consecutive measurements taken at each timestamp. For the next 45 minutes, measurements were taken at 5 minute intervals, with three consecutive measurements taken at each timestamp. Each sample was prepared in triplicate.

[0114] Example 2: Classical Pathway The solution followed the following protocol: Liposomes (in this case, with a high cholesterol content, e.g., 44 or 42 mol%) ·buffer Liposomal Complement Buffer (LCB) 10mM HEPES, 150mM NaCl, 135nM CaCl2, 1mM MgCl2, pH7.4 Inactivation buffer (for negative control, to inactivate the complement system): Liposomal Inactivated Complement Buffer (iaCB) 90% liposomal complement buffer and 200 mM EDTA, 0.5 μL EGTA pH ~8 Sucrose-containing liposome complement buffer (used in all wells to adjust the sucrose content in each well to ensure better stability of liposomes) Human serum Detergent-containing solution (used as a positive control to dissolve all liposomes) 300 mM n-octyl-β-d-glucoside (OG) in double-distilled water

[0115] Assay preparation: 1. Prepare liposome stock solution Mix the liposomes with buffer to obtain a 10x concentrated stock solution that will be needed later. 2. Prepare a black microtiter plate on ice Add buffer to all wells (volumes will vary for buffer control, activated serum, negative control, and positive control, and all wells should be 100 μL when all solutions are added at the end) Add sucrose solution (1M stock, 20 μL per well) Add inactivation buffer (10 μL to each negative control well). 3. Add detergent solution (10 μL to each positive control well). 4. Preheat the fluorescence plate reader to 37°C Steps for performing the assay: 5. Add 10 μL of liposomes (prepared in 1.) to each well. 6. Add the appropriate amount of serum to all samples containing serum (positive control, negative control, and activated serum; activated serum must be added last). The total volume of all wells must now be 100 μL. 7. Measuring time-resolved fluorescence intensity

[0116] Fluorescence intensity was measured using a BioTek SYNERGY neo2 fluorescence reader. The wavelength was λ Ex = 565(5) nm and λ Em = 585(5) nm. The measurement temperature inside the instrument was constant at 37°C. Measurements were performed with a gain of 150. Measurements were performed for 60 minutes. For the first 15 minutes, measurements were taken at 1.5 minute intervals, with three consecutive measurements taken at each timestamp. For the next 45 minutes, measurements were taken at 5 minute intervals, with three consecutive measurements taken at each timestamp. Each sample was prepared in triplicate.

[0117] Example 3: Identification of classical pathway-selective liposomes Objective: The focus of this study was to identify liposomes that could be used to identify the classical pathway.

[0118] To determine activation of the alternative pathway, EGTA, which blocks both the classical and lectin pathways, was added to the samples (see Figure 3). To distinguish between the lectin and classical pathways, several antibodies were kindly provided by Hycult Biotech and Sanquin. (i) mAb HM2285 (C3b / iC3b, human, clone 5G9) should inhibit the classical pathway but not the alternative pathway, but because it binds to C3b(i), it was unclear whether it also inhibits the lectin pathway. (ii) <anti-C1s> M81 (HM2108), provided to Hycult Biotech, binds to C1s at the C4 / C4b binding site and should therefore block C4 cleavage and further activation of the classical pathway. (iii) an <anti-C1q> antibody, designated "<anti-C1q85>," provided to Sanquin. This antibody binds to C1q in the globular head region (GR) and should effectively block the complement system, i.e., selectively block the classical pathway.

[0119] Summary of Results: We were able to demonstrate that liposomes containing high concentrations of cholesterol activate the complement system through the classical pathway (see Figures 3 and 4). This was demonstrated using three strategies: (i) the addition of EGTA did not result in liposome lysis; (ii) the addition of an antibody that blocks the classical pathway allowed us to observe an inhibition curve of complement activation, including complete inhibition at higher antibody concentrations tested; and (iii) the use of C1q-depleted serum did not result in activation of the complement system by liposomes. Compared to the red blood cell assay, which requires the addition of an antibody to specifically target the classical pathway, this is not necessary here.

[0120] Conclusion: From a broader perspective, including data not presented in this application, we are now able to perform pathway-selective complement lysis for two of the three pathways: The classical pathway can be investigated with cholesterol-rich liposomes and can also be addressed using ligand-bearing liposomes and anti-ligands, as demonstrated with biotinylated liposomes and anti-biotins. - The alternative pathway can be examined by combining LPS-containing liposomes with EGTA (to block the classical and lectin pathways). Residual lysis is an indicator of alternative pathway activity.

[0121] material

[0122] [Table 1]

[0123] Liposome Complement Buffer (LCB) 10mM HEPES, 150mM NaCl, 135nM CaCl2, 1mM MgCl2, pH7.4 Liposome Inactivated Complement Buffer (iaCB) 90% liposomal complement buffer and 200 mM EDTA, 0.5 μL EGTA pH ~8 ·antibody: mAb HM2285 (C3b / iC3b, human, clone 5G9, 100 μg / mL) mAb HM2108 (C1s, human, mAb M81, 1.02 mg / mL, Hycult) mAb <anti-C1q85> (mouse, 0.84 mg / mL, Sanquin) 1M sucrose solution in LCB Human serum C1q-depleted serum (CompTech, A300) Innovative Research Human Complement Serum Batch IR35577 Pooled serum from volunteers 1–4, 1( 1 / 6), 2( 1 / 6), 3( 1 / 3), and 4( 1 / 3) The active serum is thawed by hand, added to the wells and mixed appropriately. 300mM n-octyl-β-d-glucoside (OG) in double-distilled water

[0124] method All antibody dose-response curves were generated using the same protocol, with only the antibody being replaced.

[0125] Unless otherwise noted, antibody concentrations ranging from 0, 0.5, 1, 2, 4, and 8 μg / mL were prepared in combination with 2% IR35577 serum using 1 μL of total lipid (tL) of cholesterol-rich liposome KH210127. These values ​​represent the final concentration in the wells. For samples containing antibody, only active serum samples were measured to preserve the antibody.

[0126] Procedure (for buffer, aS, iaS, and OG controls, also prepare controls in which PBS is added instead of antibody): 1. Prepare a 10 μM (tL) liposome solution (in LCB) 2. Prepare black MTPs on ice with LCB, sucrose (1M stock in LCB, 20 µL for each well), iaCB (10 µL for each well), and 300 mM OG (10 µL for each well). 3. Add 10 μL of liposomes to each well (final concentration will be 1 μM tL per well) 4. Preheat the BioTek Reader to 37°C. 5. Mix the active serum with the respective amount of inhibitor (8 μL serum + 32 μL of the respective inhibitor, 100, 50, 25, 12.5, or 6.25 μg / mL stock). It is important that the final concentration in the well is correct. 6. Incubate the serum-inhibitor mix on ice for 30 minutes. 7. Add 10 μL of serum-inhibitor mixture to the inactive serum well and the OG well so that the serum content is 2% and the inhibitor concentration in the well is appropriate. 8. Add 10 μL of serum-inhibitor mixture to the activated serum wells so that the serum content is 2% and the inhibitor concentration in the wells is appropriate. 9. The total volume in all wells should now be 100 μL 10. Measuring time-resolved fluorescence intensity

[0127] Fluorescence intensity was measured using a BioTek SYNERGY neo2 fluorescence reader. The wavelength was λ Ex = 565(8) nm and λ Em = 585(8) nm. The measurement temperature inside the instrument was kept constant at 37°C. The measurement was performed at a gain of 150.

[0128] Variations included the <anti-C1q> antibody "anti-C1q85" tested at an updated concentration range and serum pools 1-4. <anti-C1q85> antibodies at concentrations ranging from 0, 2, 3, 4, 5, 6, 7, and 8 μg / mL were combined with 2% pooled serum 1-4 and 1 μL tL KH210127 liposomes.

[0129] Additionally, 1 μL of tL KH210127 liposomes was assayed in 2% C1q-depleted serum. To preserve the C1q-depleted serum, only active serum was assayed. The samples containing C1q-depleted serum followed the same protocol as the inhibitor solution, except that PBS was added instead of antibody. This ensured comparability between samples.

[0130] To confirm that C1q-depleted serum possesses functional complement components, high cholesterol, antibody, or LPS-modified liposomes were tested with 1 μM tL and 2% C1q-depleted serum. As a reference, normal human serum with 2% IR35577 was tested with the same liposomes.

[0131] Results mAb HM2285 (C3b / iC3b, human, clone 5G9) The antibody 5G9 blocks the complement system very effectively. The literature states that it should inhibit the classical pathway. Based on the literature, it is unclear whether it also inhibits the lectin pathway. Varying the antibody concentration from 0.5 to 8 μg / mL (in-well concentration) yielded a predicted inhibition response curve, with complete inhibition observed at 8 μg / mL of the 5G9 antibody. The results are presented as raw time course data (Figure 5) along with an associated bar graph (Figure 6) of the final fluorescence signal obtained in the time course experiment. Figure 7 provides three different data representations for analyzing the data. The relationship between antibody concentration and resulting liposome lysis is plotted, demonstrating the typical logarithmic relationship of the inhibition response.

[0132] Results <anti-C1s> antibody M81 Surprisingly, the anti-C1s antibody M81 does not affect complement activity. Although it has been reported in the literature that this antibody binds to C1s and thus blocks C4 cleavage and the classical pathway, we were unable to confirm this in actual patient samples using our liposome assay. However, it should be noted that we were unable to find a functional assay detailing the inhibitory effect of this antibody using an erythrocyte assay. The data shown include rad fluorescence time course data (Figure 8), associated bar graphs of the final endpoint fluorescence signal (Figure 9), and an inhibition curve showing lysis versus antibody concentration (Figure 10). Our liposome assay appears to adequately reflect the functionality of the complement system and is superior to complement system assays based solely on protein assays.

[0133] Results <anti-C1q85> (Sanquin), IR35577 serum The anti-C1q85 antibody was first tested with commercially available pooled human serum (IR35577). Again, a typical inhibition curve was observed at concentrations ranging between 2 μg / mL (negligible inhibition) and 8 μg / mL (complete inhibition). It should be noted that the control sample without inhibitor exhibited higher than expected lysis. Typically, approximately 70% lysis was achieved under the exact same conditions in previous experiments. Nevertheless, a clear trend was observed. The raw data are provided in Figure 11 and the associated bar graph of the fluorescent signal at the endpoint (Figure 12). The final inhibition curve, plotted as lysis versus antibody concentration, demonstrates the inhibitory potential of the antibody in this functional liposome assay (Figure 13).

[0134] Inhibition by anti-C1q85 appears to be more effective than that by 5G9 at the same concentration (4 μg / mL). These data suggest that cholesterol-rich liposomes activate only the classical pathway. Therefore, cholesterol-rich liposomes, e.g., liposomes with a cholesterol content of at least 30 mol%, can be advantageously used to specifically target the classical pathway.

[0135] Results: Anti-C1q85 (Sanquin), pooled sera 1-4, C1q-depleted sera The results showed that cholesterol-rich liposomes were induced exclusively by the classical pathway. To confirm that this was not due to irregularities in the human serum used, we performed two studies: (i) we tested these liposomes with C1q-depleted serum, and (ii) we tested these liposomes with anti-C1q85 in a slightly optimized concentration range of 2, 3, 4, 5, 6, 7, and 8 μg / mL with 2% pooled serum 1–4.

[0136] The pooled serum produced results similar to those of the previously used IR serum, but the activity range was found to be slightly shifted toward lower antibody concentrations (Figures 14, 16, 18, and 19). Furthermore, liposomes did not dissolve in C1q-depleted serum samples (Figure 15). These data demonstrate that the finding that these cholesterol-rich liposomes induce only the classical pathway is correct and is evident regardless of the serum source used, even in sera that do not activate the classical pathway.

[0137] Results C1q-depleted serum To confirm that C1q-depleted serum could indeed induce complement-induced lysis in liposomes, control experiments were performed using cholesterol-rich, antibody-, or LPS-modified liposomes. At least LPS-modified liposomes were predicted to exhibit some degree of complement-induced lysis in this serum.

[0138] This was certainly true. LPS-modified liposomes could be partially lysed by C1q-depleted serum, because the classical pathway is not the only pathway activated by this trigger. Nevertheless, C1q depletion reduced the amount of lysis compared with the positive control using normal human serum (Figures 20 and 21). This effect was also observed in studies involving LPS-modified liposomes with EGTA (not shown), indicating that the classical pathway is responsible for a significant amount of lysis even with LPS-modified liposomes.

[0139] Neither antibody-modified nor cholesterol-rich liposomes showed increased lysis in C1q-depleted serum, suggesting that only the classical pathway is activated by these triggers.

[0140] Example 4: Identification of liposomes selective for the alternative pathway Objective: The focus of this study was to identify liposomes that could be used to identify the alternative pathway. Liposomes modified with LPS in lipid membranes have been found to activate the complement system. This experiment focused on understanding whether the alternative pathway could be activated and examined separately from the other two pathways.

[0141] To determine activation of the alternative pathway, C1-Inh, a known complement inhibitor of the classical and lectin pathways, was added to the samples. 2+ than Ca 2+ EGTA, a selective complexing agent with high affinity for Ca, was investigated as a cheaper alternative to C1-Inh because the alternative pathway is to convert Ca 2+ This is because it is the only pathway that is active under conditions of starvation.

[0142] Summary of Results: We were able to demonstrate that liposomes bearing LPS on their surface partially activate the complement system through the alternative pathway. This was demonstrated by two strategies: (i) the addition of C1-Inh, which blocks both the classical and lectin pathways, reduced liposome lysis in a dose-dependent manner; (ii) Mg 2* By adding varying amounts of EGTA, we observed a complement activation inhibition curve until the residual lysis rate leveled off at the higher EGTA concentrations tested. This allowed for optimization of assays such as CH50, allowing for the interpretation of serum-specific alternative pathway complement activation. Therefore, the alternative complement pathway can be specifically targeted, which is advantageous.

[0143] Conclusion: From a broader perspective, including data not presented in this application, we are now able to perform pathway-selective complement lysis for two of the three pathways: The classical pathway can be investigated with cholesterol-rich liposomes and can also be addressed using ligand-bearing liposomes and anti-ligands, as demonstrated with biotinylated liposomes and anti-biotins. - The alternative pathway can be examined by combining LPS-containing liposomes with EGTA (to block the classical and lectin pathways). Residual lysis is an indicator of alternative pathway activity.

[0144] material

[0145] [Table 2]

[0146] Liposome Complement Buffer (LCB) 10mM HEPES, 150mM NaCl, 135nM CaCl2, 1mM MgCl2, pH7.4 Liposome Inactivated Complement Buffer (iaCB) 90% liposomal complement buffer and 200 mM EDTA, 0.5 μL EGTA pH ~8 C1 inhibitor (C1-INH) (Cinryze, manufactured by VarioPharma) 200 U / mL in sterile water 1M sucrose EGTA inactivation buffer (inactivates classical and lectin pathways) 100mM EGTA in LCB, pH7.4 Used to prepare various EGTA solutions Human serum Pooled serum 1( 1 / 6), 2( 1 / 6), 3( 1 / 3), and 4( 1 / 3) Innovative Research Human Pooled Complement Serum Thaw activated serum and add to wells. Serum was inactivated by adding 10 μL of iaCB to each well. 300mM n-octyl-β-d-glucoside (OG) in double-distilled water

[0147] method All assays were performed following the same general procedure with minor adjustments in incubation time and serum content, which are described separately for each experiment.

[0148] Tests involving C1-INH were performed with 5, 2.5, 1.25, 0.625, and 0 U / mL of C1-INH in each well, with a serum content of 5%, which translates to 0.5, 0.25, 0.125, and 0.0625 U / well (100 μL well volume) and 5 μL of serum per well.

[0149] procedure: 1. Prepare a 100 μM (tL) liposome solution 2. Prepare a dilution curve for each inhibitor stock solution 3. Mix the serum with the respective amount of inhibitor. It is important that the final concentration in the well is accurate. The dilution factor is not constant and must be calculated individually for each inhibitor. 4. Incubate the mixture for the respective time. C1-Inh: 60 minutes on ice (used only in control experiments) EGTA test: 5 minutes at room temperature (unless otherwise specified) 5. Prepare black MTPs on ice with LCB, sucrose (1 M stock, 20 µL for each well), iaCB (10 µL for each well), and 300 mM OG (10 µL for each well). 6. Add 10 μL of liposomes to each well (final 1 μM tL per 10 wells) 7. Preheat the BioTek Reader to 37°C 8. Add the appropriate amount of serum-inhibitor mixture to the inactive serum wells and OG wells to achieve the desired serum content and appropriate inhibitor concentration in the wells. 9. Add the appropriate amount of serum-inhibitor mixture to the active serum wells to achieve the desired serum content and appropriate inhibitor concentration in the wells. 10. The total volume in all wells should now be 100 μL 11. Measuring time-resolved fluorescence intensity

[0150] Fluorescence intensity was measured using a BioTek SYNERGY neo2 fluorescence reader. The wavelength was λ Ex = 565(5) nm and λ Em = 585(5) nm. The measurement temperature inside the instrument was constant at 37°C. The measurement was performed at a gain of 150.

[0151] Results: Complement assay with C1-INH using LPS-liposomes Note: Test a 5 vol% serum sample containing 10 μM tL! As can be seen in Figures 22 and 23, C1-INH inhibits LPS-liposome lysis. Lysis decreased from 45% in the absence of C1-INH to 30% at the highest inhibitor concentration investigated (5 U / mL with 5% serum). C1-INH is known to inhibit both the classical and lectin pathways. These results correlate well with this assumption, as complete inhibition was not achieved. To confirm that higher inhibitor concentrations did not result in complete inhibition, 10 U / mL C1-INH was tested in a separate experiment under the same conditions, yielding the same results as 5 U / mL (data not shown). This data suggests that LPS-liposome lysis is induced not only via the alternative pathway but also via the classical and / or lectin pathways, and blocking the latter two pathways with C1-INH reduces liposome lysis. Therefore, the lysis remaining after C1-INH addition is an indicator of alternative pathway activity for each serum.

[0152] Results: Complement assay with EGTA A less expensive but equally effective method for inactivating the classical and lectin pathways is EGTA / Mg 2+ The key to success is to use a buffer containing EGTA, which is required for the activity of the classical and lectin pathways but not the alternative pathway. 2+ The alternative pathway requires Mg 2+ That's all.

[0153] The EGTA content was varied during the 1:1 incubation with serum. After a 5-minute incubation, the serum-EGTA mixture was diluted 1 / 5 directly into the wells, resulting in EGTA concentrations of 0, 0.2, 0.4, 0.8, and 1 mM EGTA. MgCl2 was omitted from the liposome-complement buffer and included directly in the serum incubations, meaning 10 mM MgCl2 in each stock solution, 5 mM MgCl2 in the 1:1 incubation, and 1 mM MgCl2 in each well. This ensures that EGTA contributes at least an equal amount of Mg to the complex. 2+ As a control, Mg 2+ A "normal" LCB was also prepared using the sample.

[0154] The serum content in each well was 10 vol.%, and the liposome concentration was 10 μM total lipid.

[0155] Both controls without EGTA showed similar lysis of LPS liposomes, indicating that it made no difference whether MgCl2 was included during preincubation or in the wells. Advantageously, 0.4 mM EGTA was already sufficient to efficiently block the classical and lectin pathways, resulting in a lysis value of 46%. Increasing the EGTA content to as high as 1 mM EGTA (final concentration in the well) did not result in a significant inhibitory effect. Therefore, it can be inferred that the classical and lectin pathways are efficiently blocked with an EGTA content of 0.4 mM and 1 mM MgCl2.

[0156] In most of the literature, Ca 2+ Mg complexed under physiological conditions 2+ To ensure the level, Mg 2+ It was stated that the levels should be kept at the same level as EGTA. 2+ If the level is too low, the Ca 2+ in addition to the Mg provided by serum 2+ The complexing content will also leave "free uncomplexed" EGTA.

[0157] EGTA levels are related to the theoretical serum Ca 2+ level (~2.5 mM Ca 2+ A dramatic drop in lysis can be observed when EGTA concentrations equal or exceed 100% serum. Below that, the classical and lectin pathways appear to function equally well. Above that, even if only slightly, the classical and lectin pathways appear to be blocked. This is because a common buffer (liposomal complement buffer, LCB) contains only 135 nM Ca. 2+ This correlates well with the fact that only the concentration is involved. 0.4 mM EGTA was determined to be the most suitable condition for further experiments.

[0158] Results: Optimization of the complement assay using EGTA, Mg2+ Based on the above findings, various MgCl concentrations in combination with 0.4 mM EGTA (final concentration in the well) were investigated. 2+ The content varied between 0.2, 0.4, 0.6, and 1 mM MgCl in the final well. Again, EGTA was incubated 1:1 with serum. After a 5-minute incubation, the serum-EGTA mixture was diluted 1 / 5 directly into the well.

[0159] Varying the MgCl2 content without adding EGTA did not affect complement activity and resulted in complete lysis of LPS liposomes, probably because serum already contained physiological levels of MgCl2. 2+Contains high concentrations and therefore requires additional Mg for proper functioning. 2+ However, as soon as EGTA is involved, Mg 2+ Increasing levels result in increased dissolution values ​​in the concentration range investigated. Dissolution is observed at Mg below the EGTA content. 2+ The concentration of Mg in EGTA was completely blocked. 2+ The concentration of 0.4mM EGTA and 1mM Mg increases with the increase in the ratio of 2+ The greatest dissolution was observed at these conditions, which were already the most favorable in previous experiments.

[0160] In the next experiment, a more precise study will be carried out between 0.2 mM and 0.4 mM.

[0161] Results: Complement assay using EGTA, precise EGTA content test Considering the above, 1mM Mg 2+ Another detailed test was carried out on the addition of EGTA to serum before the addition of the complement assay, within a concentration range of 0.2-0.4 mM EGTA (final concentration in the well, stock solution 4 mM) with a content (final concentration in the well, stock solution 10 mM).

[0162] By narrowing the EGTA concentration range in this way, a dose-response curve could be obtained. Complete lysis was observed at 0 or 0.2 mM EGTA. At 0.25 mM EGTA, inactivation began, with lysis values ​​of approximately 90%. It is assumed that the classical and lectin pathways are only partially inactivated here. This appears to be the limit, and is within the theoretical Ca concentration in 100% serum. 2+ This would confirm that levels are approximately 2.5 mM. In contrast, 0.3 mM EGTA already appears to completely inhibit the classical and lectin pathways, as does 0.4 mM EGTA.

[0163] Results: Complement assay with EGTA, optimization of preincubation time In the example above, the optimal EGTA concentration for 1:1 preincubation with serum prior to assay addition was determined to be 0.4 mM EGTA (final concentration in the well; the stock solution was 10x concentrated). (The buffer contained 4 mM EGTA and 10 mM MgCl2 in LCB.) However, solubility varied even when working under the same concentration conditions. Therefore, we further investigated the incubation time with EGTA, varying it between 0 and 35 minutes prior to assay addition.

[0164] EGTA was mixed 1:1 with serum for various times before being added to the wells. Incubation times of 0, 5, 10, 15, 20, 25, and 35 minutes were selected. In each case, the final EGTA concentration in the well was 0.4 mM EGTA and 1 mM MgCl2. The serum content in each well was 10 vol.%. The liposome concentration was 10 μM total lipid.

[0165] The results show that within the first 20 minutes of incubation of EGTA with serum (1:1), the lysis rate drops by approximately 10%. After incubation at room temperature for up to 35 minutes, the lysis rate remains constant at approximately 42%. It is also observed that the inactivation of EGTA occurs immediately.

[0166] Overall, 0.4 mM EGTA and 1 mM Mg 2+ appears to be the most suitable condition for examining alternative pathway activity (Figures 35, 36, and 37). Inactivation of EGTA can be performed by adding the respective amount directly to the wells before adding serum, which allows for easier preparation and reproducibility of the assay.

[0167] The features of the invention disclosed in this specification, in the claims and / or in the accompanying drawings may, both individually and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. A method of determining complement activity, comprising: A) determining classical complement pathway activity; B) determining alternative complement pathway activity; and / or C) determining lectin complement pathway activity; A) determining the classical complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol; Optionally, the one or more liposomes comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; b) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; c) detecting the released marker; Including, B) determining the alternative complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise an alternative complement pathway activator, preferably lipopolysaccharide, more preferably about 0.01 mol% to about 5 mol% lipopolysaccharide, even more preferably about 1 mol% lipopolysaccharide; Optionally, the one or more liposomes comprise cholesterol, preferably ≦10 mol % cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor; Preferably, the classical complement pathway inhibitor and / or the lectin complement pathway inhibitor comprises EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP, preferably EGTA and / or C1 inhibitor; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; Including, C) determining the lectin complement pathway activity; a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; A method comprising:

2. A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and / or C) determining lectin complement pathway activity, Optionally, detecting a basal level of said marker prior to said step of contacting said one or more liposomes with said sample, at a time point ranging from about 1 second to about 20 minutes, preferably from about 1 second to about 15 minutes, after first contacting said one or more liposomes with said sample, and / or using inactivated serum and / or buffer; Optionally, determining complement activity by comparing the released markers detected in steps A) c), B) d), and C) d), respectively, with the basal level of the markers detected in the step of detecting the basal level of the markers, Preferably, a higher level of the released marker detected in steps A) c), B) d), and C) d), respectively, compared to the basal level of the marker, is indicative of complement activity. The method of claim 1 further comprising:

3. 3. The method of claim 1 or 2, wherein the detection in step A) c), step B) d), and / or step C) d) comprises or consists of time-resolved detection of the released marker.

4. 4. The method according to any one of claims 1 to 3, wherein the detecting in step A) c), step B) d) and / or step C) d) comprises or consists of time-resolved detection of the released marker, wherein an increase or decrease in the released marker over time, preferably an increase in the released marker over time, is indicative of complement activity.

5. the marker is selected from a fluorescent marker, preferably pyrenetetrasulfonic acid, sulforhodamine B, indocyanine green such as 2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, or carboxyfluorescein; an electrochemical marker, preferably potassium hexaferricyanide, potassium hexaferrocyanide, or ruthenium hexamine; a colorimetric marker, preferably sulforhodamine B; a chemiluminescent marker, preferably luminol; an electrochemiluminescent marker, preferably luminol or ruthenium bipyridyl; a bioluminescent marker such as GFP; and combinations thereof; preferably, the marker is water soluble; Preferably, the marker is selected from a fluorescent marker, an electrochemical marker, and combinations thereof; More preferably, the method according to any one of claims 1 to 4, wherein the marker is selected from sulforhodamine B and luminol.

6. the marker is a fluorescent marker, and the marker is quenched in the undissolved liposomes; Preferably, said one or more liposomes are non-lysed liposomes prior to said lysis of said one or more liposomes; Optionally, 6. The method of any one of claims 1 to 5, wherein an increase in the released markers detected in step A) c), step B) d), and / or step C) d) over time is indicative of complement activity.

7. 7. The method of any one of claims 1 to 6, wherein the one or more liposomes comprising a marker provided in step A) a) comprise a first marker, the one or more liposomes comprising a marker provided in step B) a) comprise a second marker, and the one or more liposomes comprising a marker provided in step C) a) comprise a third marker, and wherein the first marker, the second marker, and the third marker are different markers.

8. determining at least two of A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and C) determining lectin complement pathway activity, preferably simultaneously or sequentially; 8. The method according to any one of claims 1 to 7, wherein preferably said detection of said released markers of steps A) c), B) d) and / or C) d) is performed simultaneously or sequentially, preferably simultaneously.

9. determining lectin complement pathway activity; determining the lectin complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; The method according to any one of claims 1 to 8, comprising:

10. A) determining classical complement pathway activity and C) determining lectin complement pathway activity; A) determining the classical complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol; Optionally, the one or more liposomes comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; b) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; c) detecting the released marker; Including, C) determining the lectin complement pathway activity; a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; The method according to any one of claims 1 to 9, comprising:

11. B) determining alternative complement pathway activity and C) determining lectin complement pathway activity; B) determining the alternative complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise an alternative complement pathway activator, preferably lipopolysaccharide, more preferably about 0.01 mol% to about 5 mol% lipopolysaccharide, even more preferably about 1 mol% lipopolysaccharide; Optionally, the one or more liposomes comprise cholesterol, preferably ≦10 mol % cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor; Preferably, the classical complement pathway inhibitor and / or the lectin complement pathway inhibitor comprises EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP, preferably EGTA and / or C1 inhibitor; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; Including, C) determining the lectin complement pathway activity; a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; The method according to any one of claims 1 to 10, comprising:

12. A) determining classical complement pathway activity, B) determining alternative complement pathway activity, and C) determining lectin complement pathway activity; A) determining the classical complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise cholesterol, preferably at least 30 mol% cholesterol, more preferably at least 40 mol% cholesterol; Optionally, the one or more liposomes comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; b) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; c) detecting the released marker; Including, B) determining the alternative complement pathway activity a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise an alternative complement pathway activator, preferably lipopolysaccharide, more preferably about 0.01 mol% to about 5 mol% lipopolysaccharide, even more preferably about 1 mol% lipopolysaccharide; Optionally, the one or more liposomes comprise cholesterol, preferably ≦10 mol % cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and a lectin complement pathway inhibitor; Preferably, the classical complement pathway inhibitor and / or the lectin complement pathway inhibitor comprises EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP, preferably EGTA and / or C1 inhibitor; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; Including, C) determining the lectin complement pathway activity; a) providing a sample, optionally a serum sample, containing or suspected of containing one or more active complement system components, and separately providing one or more liposomes comprising a marker; the one or more liposomes comprise a lectin complement pathway activator, preferably comprising a sugar moiety, an acetyl moiety, and / or a sialic acid; Preferably, the liposomes comprise cholesterol, preferably less than 10 mol% cholesterol; b) optionally contacting the sample and / or the one or more liposomes with a classical complement pathway inhibitor and / or an alternative complement pathway inhibitor; Preferably, the classical complement pathway inhibitor comprises an anti-C1q85 antibody or an antigen-binding peptide thereof, and / or the alternative complement pathway inhibitor is selected from complement factor H, mini-complement factor H, an anti-factor B antibody or an antigen-binding fragment thereof, an anti-properdin antibody or an antigen-binding fragment thereof, and any combination thereof; c) contacting the one or more liposomes with the sample to allow the one or more activated complement system components, if present in the sample, to lyse the one or more liposomes; lysis of the one or more liposomes releases the marker from the one or more liposomes; d) detecting the released markers; The method according to any one of claims 1 to 11, comprising:

13. The one or more liposomes provided in step A)a) comprise cholesterol and phospholipids; preferably, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); more preferably, at least 30 mol % cholesterol, preferably at least 40 mol % cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 10 mol % to 30 mol %, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in the range of 25 mol % to 45 mol %; Optionally, the one or more liposomes provided in step A)a) further comprise a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; the one or more liposomes provided in step B)a) comprise phospholipids and cholesterol, preferably ≦10 mol% cholesterol; preferably 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; more preferably 65 mol% to 85 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 10 mol% to 30 mol% 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; and / or The one or more liposomes provided in step C)a) comprise phospholipids and ≦10 mol% cholesterol; preferably 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; more preferably 65 mol% to 85 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 10 mol% to 30 mol% 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and ≦10 mol% cholesterol; Optionally, the one or more liposomes provided in step A)a), step B)a), and / or step C)a) comprise 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine; 13. The method of any of claims 1 to 12, optionally wherein the one or more liposomes provided in step B) a) and / or the one or more liposomes provided in step C) a) comprise a classical complement pathway inhibitor, preferably an anti-C1q85 antibody or antigen-binding fragment thereof.

14. 14. The method according to any one of claims 1 to 13, wherein the one or more liposomes provided in step A) a), the one or more liposomes provided in step B) a), and / or the one or more liposomes provided in step C) a) are selected from anionic liposomes, PEGylated liposomes, and cationic liposomes, preferably anionic liposomes.

15. Step B)b) is Mg ２＋ in the presence of, preferably at least 0.001 mM MgCl ２ more preferably in the presence of at least 0.1 mM MgCl ２ and even more preferably at least 0.5 mM MgCl ２ , e.g., about 1 mM MgCl ２ the sample and / or the one or more liposomes in the presence of Ca ２＋ The method of any one of claims 1 to 14, comprising contacting with a complexing agent, preferably a classical complement pathway inhibitor selected from EGTA and / or a lectin complement pathway inhibitor.

16. including a positive control and / or a negative control; the positive control comprises contacting the one or more liposomes provided in steps A)a), B)a), and / or C)a) with a detergent and / or solvent, preferably a detergent, more preferably a detergent selected from n-octyl-β-d-glucoside, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polyoxyethylene(20) sorbitan monolaurate, and polyoxyethylene(80) sorbitan monooleate; 16. The method of any one of claims 1 to 15, wherein the negative control comprises replacing the sample, optionally the serum sample, in steps A)a), B)a), and / or C)a), with a negative control sample comprising or consisting of inactive serum.

17. 1. A liposome collection for determining complement activity, preferably for determining A) classical complement pathway activity, B) alternative complement pathway activity, and / or C) lectin complement pathway activity, comprising: A) a liposome for determining classical complement pathway activity, comprising a marker, cholesterol, preferably at least 30 mol % cholesterol, more preferably at least 40 mol % cholesterol, and a phospholipid; Preferably, the markers include cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); More preferably, the marker comprises at least 30 mol% cholesterol, preferably at least 40 mol% cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 10 mol% to 30 mol%, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in the range of 25 mol% to 45 mol%; Optionally, the liposome further comprises a classical complement pathway activator, preferably selected from an antibody, an antigen, and a ligand such as biotin or streptavidin; B) a liposome for determining alternative complement pathway activity, comprising a marker, an alternative complement pathway activator, cholesterol, preferably ≦10 mol% cholesterol, and a phospholipid; Preferably, the markers include an alternative complement pathway activator, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); More preferably, the marker, alternative complement pathway activator, comprises ≦10 mol% cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 65 mol% to 85 mol%, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in the range of 10 mol% to 30 mol%; Preferably, the alternative complement pathway activator is a liposome that is lipopolysaccharide, preferably from about 0.01 mol% to about 5 mol% lipopolysaccharide, more preferably about 1 mol% lipopolysaccharide; and / or C) a liposome for determining lectin complement pathway activity, comprising a marker, a lectin complement pathway activator, cholesterol, preferably ≦10 mol% cholesterol, and a phospholipid; Preferably, the markers include lectin complement pathway activator, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol); More preferably, the marker comprises a lectin complement pathway activator, ≦10 mol% cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of 65 mol% to 85 mol%, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in the range of 10 mol% to 30 mol%; Preferably, the liposome wherein the lectin complement pathway activator comprises a sugar moiety, an acetyl moiety, and / or a sialic acid; Including, Optionally, the liposome for determining alternative complement pathway activity B) and / or the liposome for determining lectin complement pathway activity C) comprise a classical complement pathway inhibitor, preferably an anti-C1q85 antibody or an antigen-binding fragment thereof; Preferably, the liposome collection comprises A) and B), A) and C), B) and C), or A), B) and C).

18. A kit for determining complement activity, comprising: - a collection of liposomes according to claim 17, a buffer, preferably a liposomal complement buffer; - optionally a classical complement pathway inhibitor, an alternative complement pathway inhibitor, and / or a lectin complement pathway inhibitor; preferably EGTA, C1 inhibitor, anti-MASP antibody, and / or C4BP; more preferably EGTA and / or C1 inhibitor; - optionally inactivated serum; preferably serum inactivated by EDTA, EGTA, heat, ultrasound and / or solvents such as water-soluble organic solvents; - optionally a surfactant, preferably n-octyl-β-d-glucoside; optionally an osmotic agent, preferably a sucrose solution; - optionally instructions for determining complement activity, preferably instructions for determining classical complement pathway activity, alternative complement pathway activity and / or lectin complement pathway activity; more preferably instructions for the method of any one of claims 1 to 12. Kit including:

19. 19. The liposome collection of claim 17 or the kit of claim 18 for use in a method for diagnosing, monitoring or treating a complement-related disorder, preferably a complement-related disease; optionally, the complement-related disease is selected from eye diseases, sepsis, kidney diseases, infectious diseases, primary complement gene deficiencies and autoimmune diseases, preferably selected from systemic lupus erythematosus, rheumatoid arthritis, hereditary angioedema, meningococcal disease, anti-factor H antibody-associated diseases, recurrent suppurative infections, hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, neuromyelitis optica disorder, atypical hemolytic uremic syndrome and C3 glomerulopathy.

Images