Novel assay for determining complement activity

Abstract

An assay method for quantifying complement activity, comprising the steps of providing a serum sample and a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance; mixing the serum sample and the liposome reagent suspension; adding a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles; activating the surface- modified liposomes; reacting the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension under conditions allowing complement activation, liposome lysis, release of the first reagent substance, and particle agglutination; and measuring the degree of agglutination turbidimetrically or nephelometrically to determine complement activity. An assay kit, computer program product; and methods for analysing and evaluating complement activity using machine learning are also disclosed.

Description

240565PC1Novel assay for determining complement activityTechnical Field

[0001] The present disclosure relates to the field of clinical diagnostic assays, in particular an assay method and an assay kit for use in complement system diagnostics and immune function testing.Background

[0002] The complement system, a crucial component of innate immunity, consists of a cascade of plasma proteins that help clear pathogens and immune complexes. Deficiencies or dysfunctions in complement components can lead to recurrent infections, autoimmune disorders, or inflammatory diseases. Clinical assays to determine complement activity are crucial for diagnosing and monitoring diseases related to the complement system, which is a part of the immune system that enhances the ability of antibodies and phagocytic cells to clear microbes and damaged cells.

[0003] The CH50 assay (50% hemolytic complement activity) is a widely used functional test to assess the integrity and activity of the classical complement pathway. The CH50 assay measures the ability of patient serum to lyse antibody-coated red blood cells (RBCs), reflecting the overall activity of the classical complement pathway. Originally, the test involved the use of sheep erythrocytes (RBCs) pre-coated with antibodies (usually anti-sheep RBC antibodies). When patient serum is added, the classical pathway is triggered, leading to the formation of the membrane attack complex (MAC) (C5b-9), which lyses the RBCs. The extent of RBC lysis is then quantified by measuring the release of hemoglobin spectrophotometrically. The CH50 value represents the dilution of serum required to achieve 50% hemolysis.240565PC2

[0004] The CH50 assay remains a fundamental tool in immunology for assessing complement function. It provides insights into immune system disorders, infections, and autoimmune conditions, making it valuable in both diagnostic and research settings.

[0005] Alternative Pathway Function (AP50 or AH50) is a test which, similarly to CH50, evaluates the function of the alternative complement pathway. AP50, however, uses rabbit red blood cells and assesses the ability of the alternative pathway to lyse these cells in the presence of magnesium-EGTA, which selectively blocks the classical pathway.

[0006] While sheep erythrocytes remain the gold standard for the classical pathway hemolytic assay (CH50), viable alternatives for assessing complement function include assays using human or rabbit erythrocytes, liposome-based platforms, and ELISA-based functional assays targeting individual pathways (classical, lectin, and alternative). Each method has its own advantages and limitations, depending on the clinical or research application.

[0007] More sensitive and specific tests, such as ELISA-based complement functional assays, turbidimetric assays, and mass spectrometry, are being developed to directly measure complement component levels and activation products (e.g., C3, C4, C5b-9). An alternative approach, involving flow cytometry- based assays, allows for a more detailed analysis of complement function at the cellular level.

[0008] One alternative to using erythrocytes is based on the use of antibody-coated liposomes containing encapsulated markers, e.g., enzymes or dyes. A homogenous, liposome-based assay for total complement activity in serum was disclosed by Donald W. Bowden etal. in 1986 (Bowden D.W., Clin.Chem., 32 / 2, 275-278, 1986). The liposomes encapsulated an enzyme, here240565PC3alkaline phosphatase, and the aqueous medium outside the liposomes contained substrate for alkaline phosphatase. When the integrity of the liposomes is damaged, the enzyme and substrate interact, resulting in a colored product, measured by its absorbance at 410 nm. The absorbance is directly related to the amount of liposome damage, which in turn is related to the amount of complement activity in the sample.

[0009] The liposome approach eliminates the variability of biological cells. Artificial liposome-based assays have been adapted for automated, high-throughput screening, e.g. by Sachiko Yamamoto et al. (Yamamoto, S., Clin. Chem., 41 / 4, 586-590, 1995) using glucose-6-phosphatase dehydrogenase (G6PDH) instead of alkaline phosphatase.

[0010] There remains a need for an improved, standardized, sensitive, and automation-compatible assay for evaluating complement activity that eliminates the variability associated with biological erythrocytes while at the same time maintaining accuracy in assessing the complement pathway function.Summary[Oil] The present disclosure relates to an assay as defined in the attached claims, incorporated herein, and addresses the need for a standardized, sensitive, and automation-compatible assay for evaluating complement activity. Additionally, it provides an improved approach for complement system analysis that may overcome limitations not previously recognized, offering advantages beyond conventional methodologies.

[0012] The invention is defined by the appended claims. The description and drawings are to be regarded as illustrative of the principles of the invention and not as limiting the scope of protection. Embodiments described herein240565PC4which do not fall within the scope of the claims are to be considered as examples useful for understanding the invention. In the event of any inconsistency between the description and the claims, the claims shall prevail.

[0013] A first aspect of the present disclosure relates to an assay method for quantifying complement activity, the method comprising the following steps, not necessarily in this order:providing a serum sample and a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance,mixing the serum sample and the liposome reagent suspension, activating the surface-modified liposomes,adding a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles,reacting the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension under conditions allowing complement activation, liposome lysis, release of the first reagent substance, and particle agglutination,measuring the degree of agglutination turbidimetrically or nephelometrically to determine complement activity.

[0014] According to an embodiment of said first aspect, the encapsulated first reagent substance is selected from small molecules, such as peptides and polynucleotides, and wherein the binding partner specific for the encapsulated first reagent substance is chosen from binding moieties, aptamers, and antibodies exhibiting binding specificity for said encapsulated240565PC5first reagent substance. Preferably said encapsulated first reagent substance presents two epitopes, same or different, available for binding by the binding partners, resulting in crosslinking and the formation of aggregates.

[0015] Preferably said encapsulated first reagent substance has a molecular weight in the interval of about 2 to about 40 kDa, preferably in the interval of about 2 to about 30 kDa, more preferably about 3 to about 20 kDa, even more preferably about 3 to about 12 kDa, and most preferably about 3 to about 6 kDa.

[0016] Thus, according to an embodiment of said first aspect, the encapsulated first reagent substance is selected from peptides and polynucleotides having a molecular weight of about 2 to about 40 kDa, preferably in the interval of about 2 to about 30 kDa, more preferably about 3 to about 20 kDa, even more preferably about 3 to about 12 kDa, and most preferably about 3 to about 6 kDa; and wherein the binding partner specific for the encapsulated first reagent substance is an antibody or mixture of antibodies selected from monoclonal or polyclonal IgA, IgG, and IgM antibodies binding specifically to the encapsulated first reagent substance.

[0017] This range ensures that the first reagent substance is released and triggers agglutination of the immunoparticles. Additionally, the choice of a low molecular weight reagent substance allows the use of a lower antigen concentration while still achieving a detectable release. For instance, in the case of ovalbumin (Mw ~ 45 kDa), a concentration of approximately 50 mg / mL of the entrapped molecule is necessary to achieve a detectable release. In contrast, for a peptide with a molecular weight of about 3000 Da, the corresponding concentration required is only about 1 - 5 mg / mL.240565PC6

[0018] Many proteins face solubility challenges at concentrations of about 50 mg / mL, along with an increased risk of aggregation and potential denaturation due to molecular crowding and altered intermolecular interactions. Additionally, as confirmed in the experimental work conducted by the present inventors, highly concentrated protein solutions tend to be viscous, making liposome extrusion for size control extremely difficult or even impractical.

[0019] According to another embodiment of said first aspect, the encapsulated first reagent substance is a multiple of, a combination, or a conjugate comprising a peptide or peptides chosen from DYKDDDDK-tag, polyglutamate-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag, NE-tag, Avi-tag, PA-tag, His-tag, and E2-tag, said multiple, combination, or conjugate comprising at least 15 amino acids. Examples of conjugates are, for example a 6xFLAG conjugate with an estimated molecular weight of 6.6 kDa, a 2 kDa PEG-MAL-3xFLAG conjugate with an estimated molecular weight of 12.3 kDa, and a 5 kDa PEG-MAL-3xFLAG conjugate with an estimated molecular weight of 18.2 kDa. Further conjugates can be made using PEG or other carrier molecules together with multiples of a chosen peptide or a combination of peptides.

[0020] According to another embodiment of said first aspect, freely combinable with any one of the aspects and embodiments disclosed herein, the surface modification of the surface-modified liposomes comprises the presence of an antigen, an antibody, or a biologically active protein bound to the surface of the liposomes.

[0021] According to an embodiment of the first aspect, the surface-modified liposomes are composed of phospholipids selected from the group consisting240565PC7of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and cholesterol.

[0022] According to a preferred embodiment of the first aspect, the surface-modified liposomes are phospholipid-based, and the surface modification comprises the presence of an antigen, an antibody, or a biologically active protein covalently bound to phosphatidyl ethanolamine in the phospholipid composition of the liposomes. Preferably an antigen is conjugated to the liposomes.

[0023] According to an embodiment, the size of the liposomes is in the range of about 100 to about 300 nm. According to yet another embodiment of said first aspect, freely combinable with any one of the aspects and embodiments disclosed herein, the liposome reagent suspension comprises a stabilizing agent.

[0024] According to yet another embodiment of said first aspect, the activation of the surface-modified liposomes is induced by addition of a binding partner with specific affinity for the antigen, antibody, or biologically active protein conjugated to the liposomes. Preferably an antigen is conjugated to the liposomes.

[0025] According to one embodiment of the above, said binding partner is added with the liposome suspension.

[0026] According to another embodiment of the above, said binding partner is present in the detection reagent suspension and the activation induced when the detection reagent suspension is added to the mixture of sample and liposome suspension.240565PC8

[0027] According to yet another embodiment of the above, said binding partner is included in a separate activation reagent added prior to the reaction step.

[0028] According to another embodiment of said first aspect, freely combinable with any one of the aspects and embodiments disclosed herein, the particles in the detection reagent suspension are coated with a binding partner specific for the encapsulated first reagent substance, and said particles are selected from the group consisting of latex particles, magnetic beads, gold nanoparticles, and microspheres.

[0029] According to yet another embodiment of said first aspect, freely combinable with any one of the aspects and embodiments disclosed herein, the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension are reacted at a temperature of 35°C to 39°C, preferably at a temperature of about 37°C.

[0030] According to yet another embodiment of said first aspect, freely combinable with any one of the aspects and embodiments disclosed herein, the agglutination is detected by nephelometric or turbidimetric measurement performed at an optical wavelength from about 400 to about 900 nm.

[0031] When the detection and quantification of the agglutination is performed by nephelometric measurement, the wavelength typically falls within the visible (400-700 nm) or near-infrared (NIR, about 800-900 nm) spectrum. The choice of wavelength is influenced by the scattering properties of the sample, the detection system, and the need to minimize background interference. In order to optimize detection in protein-based and immunoassay applications, it is recommended to operate in the infrared (about 800 nm) range.240565PC9

[0032] When the detection and quantification of the agglutination is performed by turbidimetric measurement, the wavelength may also fall within the visible (400-700 nm) or near-infrared (NIR, about 800-900 nm) spectrum, depending on sample composition, particle size, and optical properties. Operating in the near-infrared spectrum helps to reduce interference from colored samples and also helps avoid background absorption in biological fluids such as serum and plasma. Within the visible spectrum, "blue light" or shorter wavelengths (about 400 - 500 nm) are preferably used for detecting smaller particles due to Rayleigh scattering, which increases with decreasing wavelength. Similarly, "green light" or wavelengths of about 540 - 580 nm are often used in protein-based assays, balancing scattering efficiency and sample transparency. "Red light" or slightly longer wavelengths (about 600 - 700 nm) are preferably used when there is a need for reducing interference from other sample components, as longer wavelengths scatter less.

[0033] A second aspect of the present disclosure relates to an assay kit for determining complement activity, comprising:a liposome reagent suspension comprising surface-modified liposomes encapsulating a first reagent substance,a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance,a reagent capable of activating the surface-modified liposomes, and one or more buffer solutions for maintaining assay conditions.

[0034] The composition, such as the character and molecular weight of the reagents is as defined in relation to the method according to the first aspect.240565PC10

[0035] A third aspect relates to a computer program product comprising computer-executable instructions which, when executed on a processor, cause a computing device to perform a method comprising the following steps:providing a serum sample and a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance,mixing the serum sample and the liposome reagent suspension, activating the surface-modified liposomes,adding a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles,reacting the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension, under conditions allowing complement activation, liposome lysis, release of the first reagent substance, and particle agglutination, andmeasuring the degree of agglutination turbidimetrically or nephelometrically to determine complement activity,wherein the program is configured to:receive assay data corresponding to the degree of agglutination measured turbidimetrically or nephelometrically,process the assay data to quantify complement activity, and output a quantified complement activity value or a corresponding diagnostic result.240565PC11

[0036] A fourth aspect of the present disclosure relates to a method for analysing and evaluating complement activity using machine learning, the method comprising:performing the assay method according to the first aspect or any embodiment thereof, to obtain assay data indicative of complement activity, inputting the assay data into a trained machine learning model configured to correlate assay signals with complement activity levels, processing the assay data using the machine learning model to generate a quantified value or classification of complement activity status, andoutputting the quantified value or classification result.

[0037] According to an embodiment of said fourth aspect, the machine learning model is trained using a dataset comprising historical assay results and corresponding clinical diagnoses.

[0038] According to another embodiment of said fourth aspect, freely combinable with the above, the step of processing the assay data using the machine learning model comprises anomaly detection for identifying atypical complement activity profiles.

[0039] A fifth aspect of the present disclosure relates to a system for determining complement activity, comprising:a sample handling unit configured to mix a serum sample with a liposome reagent suspension and a detection reagent suspension,a reaction module configured to maintain reaction conditions allowing complement activation, liposome lysis, release of a first reagent substance, and particle agglutination,240565PC12a detection module comprising a turbidimeter or nephelometer for measuring the degree of agglutination,a processing unit configured to analyze the detected signal and determine complement activity,a user interface for displaying the result.

[0040] A sixth aspect relates to a method for remotely analysing complement activity assay data, the method comprising:receiving assay data generated by the method according to the first aspect or any embodiment thereof, from a remote laboratory device, transmitting the assay data to a cloud-based server for processing, applying a trained machine learning model to process the assay data and determine a quantified value,transmitting the processed complement activity result back to the remote laboratory device for display or further analysis.Short description of the drawings

[0041] The invention is now described, by way of example, with reference to the accompanying drawings, in which:

[0042] Figure 1 schematically shows a liposome 1 containing an encapsulated first reagent substance 2, and an antigen 3 on its surface (Panel A). To activate the complement system, a polyclonal or monoclonal antibody 4 is added, the antibody binding specifically to the antigen 3. When the complement system is activated 4, this ultimately results in the formation of a membrane attack complex 5, and the release of the encapsulated reagent substance 2 (Panel B). The concentration of released reagent 2 reflects the total complement activity in the serum samples and is detected as increasing240565PC13agglutination with increasing complement activation. The signal is enhanced by using immunoparticles coated with antibodies to the encapsulated antigen and can be both turbidimetrically and nephelometrically detected (Panel C).

[0043] Figure 2 is a schematic overview of assay parameters used when performing the claimed assay on an automated clinical chemistry analyzer.

[0044] Figure 3 shows the calibration curves using the standards described in Table 2 and the liposome reagent based on phospholipid composition 1 (POPC) and phospholipid composition 2 (DPPG).

[0045] Figure 4 shows the calibration curve of an inventive 3xFLAG-based assay on an automated turbidimetric clinical chemistry analyzer.

[0046] Figure 5 shows the calibration curve of an inventive 3xFLAG-based assay on an automated nephelometric clinical chemistry analyzer.

[0047] Figures 6, 7 and 8 schematically illustrate how the binding partner (*) for activating the liposomes can be included in or added together with the liposome suspension (Fig. 6), with the detection reagent suspension (Fig. 7), or as a separate reagent solution (Fig. 8).

[0048] Figure 9 is a graph showing the dose response relationship obtained in Example 12 using liposome reagents containing different conjugates, 3xFLAG, 6xFLAG, 2k-PEG-3XFLAG, and 5k-PEG-3xFLAG.

[0049] Figure 10 is a bar diagram showing the recovery release (delta AU 10% deoxycholate / delta AU serum 50 U / ml) for 4 different conjugates:3xFLAG, 6XFLAG, 2k-PEG-3xFLAG, and 5k-PEG-3xFLAG.Description

[0050] Before the present invention is described, it is to be understood that the terminology employed herein is used for the purpose of describing240565PC14particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. Additionally, unless otherwise indicated, the following terms as used herein are to be understood in accordance with the definitions set out below.

[0051] First, it must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] As used herein, the term "about" in relation to a numerical value denotes a variation of ±10% of that value, unless a different meaning is explicitly stated or would be understood by the skilled person in the specific technical context.

[0053] As used herein, "complement activity" refers to the functional ability of the classical complement pathway to mediate lysis, measured as the degree of liposome lysis induced by the serum sample in a predefined timeframe. In this context, "complement activation" refers to the triggering of the classical complement pathway, leading to the sequential activation of complement proteins, formation of the membrane attack complex (MAC), and subsequent lysis of the liposomes or other target particles.

[0054] "Liposome reagent suspension" refers to an aqueous suspension of phospholipid-based liposomes that encapsulate a first reagent substance and are designed to undergo complement-mediated lysis, thereby releasing the encapsulated first reagent substance into the surrounding medium.

[0055] "First reagent substance" refers to a detectable marker compound encapsulated within the liposomes, which is released upon complement-mediated liposome lysis. This substance is a small molecule capable of being240565PC15detected via binding-induced particle agglutination, such as a peptide or polynucleotide.

[0056] "Detection reagent suspension" refers to a suspension containing particles (such as latex particles of a size in the range of about 100 to about 400 nm, preferably about 150 to about 300 nm) coated with a binding partner specific for the encapsulated first reagent substance, wherein binding of the released first reagent substance induces particle agglutination that is detectable turbidimetrically or nephelometrically.

[0057] "Phospholipid-based liposomes" are lipid vesicles formed from bilayered phospholipid molecules that may include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, cholesterol, or other lipid components to ensure structural stability and complement activation properties.

[0058] "Second reagent substance" refers to a molecular entity conjugated to the external surface of the liposomes that can facilitate targeted complement activation through interaction with an added antibody or binding molecule.

[0059] "Turbidimetric measurement" refers to the quantification of complement activity based on changes in optical density due to particle agglutination, whereas "nephelometric measurement" refers to lightscattering detection of agglutinated particles at an angle relative to the incident light source.

[0060] "Processing unit" refers to a computational device or system configured to analyze detected signals from the assay, quantify complement activity, and provide a numerical or categorical output based on predefined analytical algorithms.240565PC16

[0061] "Artificial intelligence model" refers to a computational system capable of analyzing assay data, detecting anomalies, and generating predictive insights related to complement activity based on prior training with clinical datasets.

[0062] "System for quantifying complement activity" refers to an integrated apparatus comprising components for sample handling, incubation, detection, data processing, and user interface interaction to measure CH50 values in a controlled laboratory setting.

[0063] "User interface" refers to a graphical or digital display system that presents assay results, trends, and diagnostic insights to the end user in an interpretable format.

[0064] "Remote analysis" refers to the process of receiving, transmitting, and processing assay data over a network, wherein analysis may be performed on a cloud-based server or distributed computing system, with results transmitted back to the originating laboratory device.An assay method applicable to either turbidimetric or nephelometric analyzers

[0065] A first aspect of the present disclosure relates to an assay method for quantifying complement activity, the method comprising the following steps: - providing a serum sample and a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance, - mixing the serum sample and the liposome reagent suspension,- adding a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles,240565PC17- activating the surface-modified liposomes,- reacting the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension under conditions allowing complement activation, liposome lysis, release of the first reagent substance, and particle agglutination,- measuring the degree of agglutination turbidimetrically or nephelometrically to determine complement activity.

[0066] According to an embodiment of said first aspect, the encapsulated first reagent substance is selected from small molecules, such as peptides and polynucleotides, and wherein the binding partner specific for the encapsulated first reagent substance is chosen from binding moieties, aptamers, and antibodies exhibiting binding specificity for said encapsulated first reagent substance.

[0067] Preferably said encapsulated first reagent substance has a molecular weight in the interval of about 2 to about 40 kDa, preferably in the interval of about 2 to about 30 kDa, more preferably about 3 to about 20 kDa, even more preferably about 3 to about 12 kDa, and most preferably about 3 to about 6 kDa. A low molecular weight is advantageous as it allows the loading of the liposomes with a greater number of said first reagent substance. It is however important that said encapsulated first reagent substance comprises at least two binding sites of epitopes, allowing simultaneous binding of at least two binding partners, e.g. aptamers or antibodies, to each reagent substance molecule.

[0068] Another consideration, if the encapsulated first reagent substance is a human biomarker, is that it is preferably chosen from biomarkers which at their highest concentration are present only in concentrations below 50 ng / L240565PC18in order not to interfere with the assay. Preferably the encapsulated first reagent substance is chosen from non-human substances.

[0069] The claimed assay and embodiments thereof have multiple advantages. By using liposomes, the variability in erythrocyte susceptibility to complement-mediated lysis, for example due to variability in R.BC batches, is avoided. The use of stable liposome formulations simplifies reagent handling and reduces costs associated with biological sample variability. Also, by reducing the number of manual steps, the claimed assay offers improved reproducibility and a greater potential of automation.

[0070] Importantly, the claimed assay can be performed with both turbidimetric and nephelometric detection, facilitating integration into automated clinical analyzers. Turbidimetry measures a reduction in the intensity of light as it passed through a sample, whereas nephelometry measures the light scattered by the particles at an angle to the incident light. Thus, in a nephelometric instrument, the detector is placed to the side, often at an angle of 90 degrees, whereas in a turbidimetric instrument, the detector is in line with the light source. It is a surprising advantage that the same assay can be run on instruments of different types, i.e. instruments operating by two different measurement principles.

[0071] The use of a first reagent substance and specific antibodies for detection allows for highly sensitive and specific detection. Similarly, the different biomarker options enable customization of the assay for various diagnostic needs, broadening its clinical applicability.An assay kit

[0072] A second aspect of the present disclosure relates to an assay kit for determining complement activity, comprising:240565PC19a liposome reagent suspension comprising surface-modified liposomes encapsulating a first reagent substance,a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance,a reagent capable of activating the surface-modified liposomes, and one or more buffer solutions for maintaining assay conditions.

[0073] An assay kit for use in automated clinical analyzers is a prepackaged set of reagents designed to enable the quantitative or qualitative detection of specific analytes in biological samples, such as blood, serum, plasma, urine, or other bodily fluids. Specifically, the kit contains a primary reactant (Rl), in this case a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance, and a secondary reagent (R2), in this case a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles. The kit may also contain calibration and control solutions. A calibrator is used to establish reference standards, while a control solution is used to verify assay accuracy and precision.

[0074] The kit may also comprise one or more buffers and / or dilution solutions to ensure optimal pH, ionic strength, and reaction conditions for the assay to function properly.

[0075] The reagents are provided in ready-to-use cartridges, vials, or bottles, designed for direct loading into clinical turbidimetric or nephelometric analyzers (for example, but not limited to the AU and DxC series from BECKMAN COULTER, the BC and BS series from MINDRAY, the CobasRproduct range from ROCHE, the Architect C-series and Alinity C-series from240565PC20ABBOT, the BNTMII product range or the AtellicaRNEPH product range from SIEMENS HEALTHINEERS). An assay kit according to this aspect and embodiments thereof is thus optimized for seamless integration into automated platforms, ensuring high-throughput, reproducibility, and minimal manual intervention.

[0076] The composition of the reagents is as defined in relation to the method according to the first aspect.

[0077] The binding partner which activates the liposomes can be included in or added together with either the liposome suspension or the detection reagent suspension, or added separately, as schematically illustrated in Fig.6, 7 and 8. Thus, according to one embodiment of the above, said binding partner is added together with the liposome suspension. According to another embodiment, said binding partner is present in the detection reagent suspension and the activation induced when the detection reagent suspension is added to the mixture of sample and liposome suspension.According to yet another embodiment of the above, said binding partner is included in a separate activation reagent added prior to the reaction step. The inventors found that the liposome suspension is more stable and thus has a longer shelf-life when the binding reagent is provided separately, either as part of the detection reagent suspension, or as a separate activation reagent.A computer program

[0078] A third aspect relates to a computer program product comprising computer-executable instructions which, when executed on a processor, cause a computing device to perform a method comprising the following steps, however not necessarily in this order:240565PC21providing a serum sample and a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance,mixing the serum sample and the liposome reagent suspension, adding a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles,activating the surface-modified liposomes,reacting the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension, under conditions allowing complement activation, liposome lysis, release of the first reagent substance, and particle agglutination, andmeasuring the degree of agglutination turbidimetrically or nephelometrically to determine complement activity,wherein the program is configured to:receive assay data corresponding to the degree of agglutination measured turbidimetrically or nephelometrically,process the assay data to quantify complement activity, and output a quantified complement activity value or a corresponding diagnostic result.A method for analysing and evaluating complement activity using machine learning

[0079] A fourth aspect of the present disclosure relates to a method for analysing and evaluating complement activity using machine learning, the method comprising:240565PC22performing the assay method according to the first aspect or any embodiment thereof, to obtain assay data indicative of complement activity, inputting the assay data into a trained machine learning model configured to correlate assay signals with complement activity levels, processing the assay data using the machine learning model to generate a quantified value or classification of complement activity status, andoutputting the quantified value or classification result.

[0080] According to an embodiment of said fourth aspect, the machine learning model is trained using a dataset comprising historical assay results and corresponding clinical diagnoses.

[0081] According to another embodiment of said fourth aspect, freely combinable with the above, the step of processing the assay data using the machine learning model comprises anomaly detection for identifying atypical complement activity profiles.A system for determining complement activity

[0082] A fifth aspect of the present disclosure relates to a system, preferably an automated system, for determining complement activity, comprising: a sample handling unit configured to mix a serum sample with a liposome reagent suspension and a detection reagent suspension,a reaction module configured to maintain reaction conditions allowing complement activation, liposome lysis, release of a first reagent substance, and particle agglutination,a detection module comprising a turbidimeter or nephelometer for measuring the degree of agglutination,240565PC23a processing unit configured to analyse the detected signal and determine complement activity,a user interface for displaying the result.A method for remotely analysing complement activity assay data

[0083] A sixth aspect relates to a method for remotely analysing complement activity assay data, the method comprising:receiving assay data generated by the method according to the first aspect or any embodiment thereof, from a remote laboratory device, transmitting the assay data to a cloud-based server for processing, applying a trained machine learning model to process the assay data and determine a quantified value,transmitting the processed complement activity result back to the remote laboratory device for display or further analysis.

[0084] The claimed computer program product enables automated processing of assay data, thus reducing manual interpretation errors. Additionally, machine learning-based analysis enhances result accuracy and enables anomaly detection for improved diagnostic reliability. Finally, remote analysis via cloud-based platforms supports telemedicine and decentralized laboratory testing.Examples

[0085] The invention is further illustrated by the following examples, which demonstrate embodiments of the claimed invention. These examples are provided solely for illustrative purposes and are not intended to limit the scope of the invention. It will be understood by those skilled in the art that240565PC24various modifications, substitutions, and adaptations may be made without departing from the spirit and scope of the invention, as defined by the appended claims. The invention encompasses all variations, equivalents, and alternatives that fall within the scope of the claims.Example 1. Choice of encapsulated first reagent substance

[0086] One possible reagent to be encapsulated by the liposomes is the DYKDDDDK-tag (FLAG®-tag, Sigma-Aldrich Co. LLV), preferably multiples and / or conjugates thereof, such as, but not limited to 3*FLAG®. This is a commonly used epitope or peptide tag that is not derived from a natural protein. The FLAG®-tag is an artificial tag and, based on its peptide sequence, it is also called a DYKDDDDK-tag. This can be used to tag proteins for multiple capture and detection applications.

[0087] The FLAG®-tag contains 8 amino acids (aa) and has the sequence motif NH2-DYKDDDDK-COOH (Asp-Tyr-Lys-Asp-. Asp-Asp-Asp-Lys). The sequence contains an enterokinase cleavage site (DDDDK), so cleavage by enterokinase easily removes the FLAG®-tag from the tagged protein. In addition to l*FLAG®-tag, 3*FLAG®-tag is also commonly used. The most frequently used sequence motif is not just a simple repetition of the 1*FLAG sequence: DYKDHDG-DYKDHDI-DYKDDDDK. The l*FLAG®-tag comprises 8 amino acids and has a molecular weight (MW) of about 1 kDa. The 3*FLAG®-tag comprises 22 amino acids and has a molecular weight (MW) of about 3 kDa. In the present context, 1*FLAG® is not sufficient, as the encapsulated reagent should present at least two binding sites (same or different) for the binding partners, e.g. the detection antibodies.

[0088] In addition to the above, the marker can be a multiple or a combination of a peptide or peptides chosen from polyglutamate-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag, NE-tag, Avi-tag, PA-tag, His-tag,240565PC25and E2-tag, said multiple or combination comprising at least 15 amino acids. In the present examples, 3*FLAG and conjugates of 3*FLAG were used.Example 2. Preparation of liposomes

[0089] Phospholipids, such as DMPC, POPC, DNP-cap-PE (16:0 DNP-cap-PE), and cholesterol, were dissolved in chloroform at suitable concentrations. DPPG and PS were dissolved in chloroform with an addition of methanol.

[0090] Three different phospholipid compositions, as outlined in Table 1, were used. An aliquot of phospholipid, such as about 10 to 15 mg, preferably 11 to 12 mg of phospholipid, with the molar composition specified in Table 1, was added to a round-bottom flask. Nitrogen gas was introduced into the flask through a rubber septum, and the solvent was evaporated under a stream of nitrogen gas while the flask was continuously rotated to form a thin lipid film on the glass walls. To remove traces of solvent, the film was further dried under a stream of nitrogen gas.Table 1: Description of phospholipid composition 1, 2 and 3 used in liposome preparationLipid Phospholipid Phospholipid PhospholipidComposition Composition 2 Composition1 [mol%] [mol%] 3 [mol%]DMPC (14:0 44.6 44.6 44.6PC)PS - - 5.0POPC 5.0 - - (16:0-18:1PC)DPPG - 5.0 - Cholesterol 49.5 49.5 49.5DNP-cap-PE 1.0 1.0 1.0(16:0 DNP-cap-PE)240565PC26

[0091] After drying, the lipid film was hydrated by the addition of 1 mL of ovalbumin solution at a concentration of 50 mg / mL, dissolved in a buffer comprising TRIS, NaCI, ProCiin™ 950, adjusted to pH 7.8. Alternatively, the lipid film was hydrated by the addition of 3*FLAG, 6*FLAG or 3*FLAG conjugates of branched 4-armed PEG-maleimide of different molecular weights (for example 2 kDa PEG-MAL-3xFLAG and 5 kDa PEG-MAL- 3 x FLAG), dissolved in a buffer comprising TRIS, NaCI, and ProCiin™ 950, at pH 7.8. The hydration process was carried out by incubating the mixture in a water bath at 35 °C for 10 minutes, followed by vortexing at high rpm. This process was repeated until all phospholipids were fully suspended in the protein solution. The resulting liposomes were then allowed to continue hydration overnight at 4 °C.

[0092] After the hydration step, the liposomes were sequentially extruded through polycarbonate membranes of varying pore sizes to achieve the desired size of 200 nm.

[0093] Excess protein or peptide, which was not entrapped within the liposomes, was removed by size-exclusion chromatography (SEC) using a column packed with Sepharose™ 4B (Sigma-Aldrich).

[0094] Absorbance at 340 nm was measured for the final liposome preparation before dilution with the complement reaction buffer to achieve an optical density (OD) of approximately 4 mAU at 340 nm.Example 3. Preparation of liposome reagent in complement buffer (Rl)

[0095] Liposomes with Ovalbumin were diluted in a buffer containing Tris, NaCI, CaC , MgCh, heat-inactivated serum, sucrose, and ProCiin™ 950, adjusted to pH 7.8. Liposomes were diluted in buffer to achieve a final OD of 4 mAU at 340 nm. The activation antibody can be mixed with the liposome240565PC27reagent, with the particle reagent or formulated as a separate reagent.When mixed with the liposome reagent, polyclonal rabbit anti-DNP was used at a final concentration of 25 pg / mL.

[0096] Liposomes with 3*FLAG and conjugates of 3*FLAG (6*FLAG or 3*-FLAG conjugates of 4-armed PEG-maleimide of different molecular weights) was diluted in a buffer containing Tris, NaCI, NZ-amine, ProCiin™ 950, adjusted to pH 7.8. The initial 1-mL-liposome preparation was diluted to a final volume. The activation antibody was either mixed with the particle reagent at a concentration of approximately 12-80 pg / mL or formulated as a separate reagent at a concentration of 1 - 10 pg / mL. Depending on the choice of antigen and corresponding activation antibody, the concentration can vary between 1 and 100 pg / mL. In the examples presented herein, the concentration was 1.4 pg / mL.Example 4. Preparation of immunoparticles (R2)

[0097] Latex particles (250-300 nm in size) bearing carboxyl (-COOH) groups were diluted to a final concentration of 0.5% in a coupling buffer containing MES and NaCI (pH 6.0). The particles were mixed at room temperature and then centrifuged at 18,000xg, the supernatant was discarded, and the pellet was resuspended in coupling buffer and sonicated.

[0098] Subsequently, 2 mM EDC and 5 mM sulfo-NHS were added to the suspension, which was incubated at room temperature. Following incubation, the suspension was centrifuged again under the same conditions, the supernatant was discarded, and the pellet was resuspended in coupling buffer and sonicated.

[0099] Antibodies specific to ovalbumin or 3*FLAG were added to the suspension of latex particles. The mixture was incubated at room240565PC28temperature to allow coupling. After antibody coupling, the reaction was quenched by adding BSA to a final concentration of 1 mg / mL, followed by incubation at room temperature. In alternative preparations of the immune-particle reagent, BSA in the quench buffer was replaced with casein (1 mg / mL), gelatin (1 mg / mL), or ethanolamine (1% v / v).

[0100] The suspension was centrifuged at 18,000xg, the supernatant was discarded, and the pellet was resuspended in a wash buffer composed of TRIS, NaCI, glycine, Tween 20, and ProCiin™, adjusted to pH 8.1. The suspension was sonicated, mixed at room temperature, and centrifuged again under the same conditions. This wash step was repeated twice.

[0101] After washing, the pellet was resuspended in a storage buffer containing Tris, NaCI, CaCl2, MgCh, BSA, sucrose, Tween 20, and ProCiin™ adjusted to pH 7.8. The suspension was sonicated and diluted to a final latex concentration of 0.1% in the storage buffer. The solution was matured at 37°C. After maturation, it was sonicated and cooled to 2-8°C for storage.

[0102] In an alternative conjugation protocol, latex particles (301 nm in size) bearing chloromethyl (-CH2CI) groups were diluted to a final concentration of 0.5% in a HEPES saline buffer (HEPES, NaCI, adjusted to pH 7.4). The particles were mixed at room temperature and centrifuged at 18,000xg, the supernatant was discarded, and the pellet was resuspended in a coupling buffer containing carbonate and NaCI (pH 9.5), followed by sonication. Antibodies specific to ovalbumin or 3*FLAG was added at a suitable concentration of antibody to latex, and the suspension was incubated at room temperature to allow antibody coupling.

[0103] After coupling, the suspension was centrifuged at 18,000xg, and the supernatant was discarded. The pellet was resuspended in a quench buffer consisting of glycine and BSA or Ovalbumin for 3*FLAG (pH 7.0). The240565PC29mixture was sonicated, mixed at room temperature, and centrifuged again under the same conditions.

[0104] The resulting pellet was resuspended in a wash buffer containing HEPES, NaCI, Tween 20, and BSA, adjusted to pH 8.1, followed by sonication and mixing at room temperature. This wash step was repeated twice. After the final centrifugation at 18,000xg, the pellet was resuspended in a storage buffer composed of Tris, NaCI, Glycine, Tween 20, and ProCiin™ at pH 8.8. The suspension was matured at 37°C and subsequently diluted to a final latex concentration of 0.1% in storage buffer.Example 5. Preparation of 3*FLAG conjugates

[0105] A 3xFLAG-Cys peptide was dissolved in coupling buffer to a concentration of 5 mg / mL. Disulfide bonds were reduced by adding TCEP to a final concentration of 1 mM and incubating the solution for 20 minutes at room temperature. TCEP was subsequently removed by desalting using PD Miditrap G-10 columns, and the reduced 3xFLAG-Cys peptides were eluted in a total volume of 2 x 1.2 mL coupling buffer.

[0106] Dimerization of 3xFLAG-Cys to formation of 6*FLAG was carried out using the homobifunctional maleimide crosslinker tetramethylenedimale-imide (BMB). BMB was freshly dissolved in dry DMSO to a concentration of 50 mM and added dropwise to the peptide solution while mixing to obtain a molar ratio of peptide to crosslinker of approximately 2:1. The reaction mixture was incubated for 60 minutes at room temperature. After completion of the reaction, remaining maleimide groups were quenched by addition of L-cysteine to a final concentration of 5 mM followed by incubation for 10 minutes.240565PC30

[0107] For PEG conjugation reactions, both 4-armed branched PEG-maleimide (PEG-MAL 2 kDa, and PEG-MAL 5 kDa) and peptide were dissolved in coupling buffer (pH 6.8-7.0). A 3xFLAG-Cys peptide was reduced with TCEP and subsequently desalted to remove the reducing agent prior to conjugation. PEG-MAL solutions were prepared at concentrations of approximately 20 mg / mL, corresponding to 10 mM for 2 kDa PEG-MAL and 4 mM for 5 kDa PEG-MAL. Conjugation reactions were performed by adding reduced 3xFLAG-Cys peptide to PEG-MAL to obtain a substitution ratio corresponding to four equivalents of peptide per PEG molecule. The reaction mixtures were incubated for 60 minutes at room temperature with gentle mixing to allow thiol-maleimide coupling. Residual maleimide groups were subsequently quenched by addition of L-cysteine to a final concentration of 5 mM followed by incubation for 10-15 minutes.

[0108] The apparent molecular weight of the PEG-FLAG conjugates was determined by size exclusion chromatography (SEC) using globular protein standards. A calibration curve was generated by plotting the logarithm of the molecular weight against the elution volume of the standards, and the elution volume of each conjugate peak was used to estimate its apparent molecular weight from the calibration curve. Based on the elution volumes, the apparent molecular weight of the 6xFLAG conjugate was estimated to be 6.6 kDa, the 2 kDa PEG-MAL-3xFLAG conjugate 12.3 kDa, and the 5 kDa PEG-MAL-3xFLAG conjugate 18.2 kDa. Ovalbumin (45 kDa) was included as a reference protein.Example 6. Assay parameters for an automated clinical chemistry analyzer -Ovalbumin based assay

[0109] The liposome reagent and immunoparticle reagent described above were implemented on an automated clinical chemistry analyzer240565PC31(Mindray BS-240), which operates on the turbidimetric detection principle. The assay parameters are schematically shown in figure 2. The assay was programmed as follows:

[0110] Addition of Liposome Reagent with activation antibody(Rl): 200 pL of liposome reagent (Rl) was added to the reaction mixture.

[0111] Addition of Serum Sample: 5 pL of serum sample was added for Rl based on liposome composition 1 (POPC) and 2 pL sample for Rl based on liposome composition 2 (DPPG).

[0112] Incubation: The mixture was incubated at 37 °C for approximately 10 minutes to allow the complement reaction to occur, resulting in the release of ovalbumin.

[0113] Addition of immunoparticles (R2): 20 pL of immunoparticles (R2) was added, and the blank measurement was taken at a pre-determined time, for example approximately 45 seconds later.

[0114] Absorbance Reading: Absorbance at 546 nm was measured over a pre-determined time period, for example 10 minutes.

[0115] The assay was configured as an endpoint reaction, measuring the total change in absorbance from the blank (taken approximately 45 seconds after R2 addition) to the end of the reaction (approximately 10 minutes after R2 addition). The selected unit for the assay was U / mL which is the standard unit for liposome-based assays evaluating complement function.240565PC32Example 7. Calibration of the assay on an automated clinical chemistry analyzer (Mindray BS-240)

[0116] A set of standards were prepared based on human serum from a healthy donor. The concentration of each standard in U / mL was measured using a commercial liposome-based assay for total complement function, Autokit CH50 from FUJIFILM Wako Chemicals. Autokit CH50 uses a homogeneous population of small-size liposomes (200 nm). The measured concentrations are presented in table 2.Table 2: Assigned concentration for standards used for calibration Standard Assigned cone. [U / mL]CAL 1 0,28CAL 2 6,32CAL 3 26,96CAL 4 47,40

[0117] The assays based on phospholipid composition 1 (POPC) and phospholipid composition 2 (DPPG) were calibrated based on triplicate measurement of each standard. The resulting calibration curves are shown in figure 3.Example s. Measurement of samples

[0118] Four samples were measured in triplicate with the commercial assay Autokit CH50 (FUJIFILM Wako Chemicals) (Table 3), the assay based on phospholipid composition 1, POPC (Table 4), and the assay based on phospholipid composition 2, DPPG (Table 5).240565PC33Table 3: Results for measurement of samples in triplicate using the commercial reagent kit Autokit CH50, FUJIFILM Wako Chemicals Sample Mean [U / mL] CV [%]Low control WAKO 18.7 3.0Normal control WAKO 47.8 2.3Serum 1 59.0 1.6Serum 2 47.0 0.4Table 2: Results for measurement of samples in triplicate using the liposome reagent based in phospholipid composition 1 (POPC). Also shown is recovery compared to measured concentration with Autokit CH50, FUJIFILM Wako ChemicalsMeanSample [U / mL] CV [%] Recovery [%] vs Autokit CH50 Low controlWAKO 14.7 5.2 79Normal controlWAKO 56.5 2.2 118Serum 1 53.6 0.7 91Serum 2 50.0 1.2 106Table 3: Results for measurement of samples in triplicate using the liposome reagent based in phospholipid composition 2 (DPPG). Also shown is recovery compared to measured concentration with Autokit CH50, FUJIFILM Wako ChemicalsMeanSample [U / mL] CV [%] Recovery [%] vs Autokit CH50 Low controlWAKO 11.3 33.1 60240565PC34Normal controlWAKO 42.6 7.7 89Serum 1 42.8 3.6 72Serum 2 39.2 0.2 83

[0119] The reagent formulation based on phospholipid composition 1 (POPC) results in better agreement with Autokit CH50 and lower CV% for samples covering the low to normal range of complement activity. A comparison shows that both new assays gave results in acceptable agreement with the commercial assay in the normal range.Example 8. Assay parameters for the turbidimetric analyzer Mindray BS-240 and the 3* FLAG based assay

[0120] The liposome reagent was prepared according to the description above using phospholipid composition 3. The particle reagent was composed of anti-FLAG coated particles at 0.1% and anti-DNP at a concentration of 80 pg / mL in a buffer containing Tris, NaCI, Glycine, Tween 20, and Proclin 950, at pH 8.8. The reagents were used on the chemistry analyzer Mindray BS240. The assay was programmed as follows:

[0121] Addition of Liposome Reagent (Rl): 130 pL of liposome reagent (Rl) was added to the reaction mixture.

[0122] Addition of Serum Sample: 7 pL of serum sample was added.

[0123] Incubation: The mixture was incubated at 37 °C for approximately 3 minutes.

[0124] Addition of immunoparticles (R2): 40 pL of immunoparticles with activation antibody (R2) was added, and the blank measurement was taken at a pre-determined time, for example approximately 30 seconds later.240565PC35

[0125] Absorbance Reading: Absorbance at 546 nm was measured over a pre-determined time period, for example approximately 10 minutes.

[0126] The assay was configured as an endpoint reaction, measuring the total change in absorbance from the blank (taken approximately 45 seconds after R2 addition) to the end of the reaction (approximately 10 minutes after R2 addition). The selected unit for the assay was U / mL which is the standard unit for liposome-based assays evaluating complement function.Example 9. Calibration and measurement of samples

[0127] The assay using the assay parameters below was calibrated using a single calibrator with the assigned concentration of 55 U / mL. The calibrator was diluted in the ratios 1:2.5, 1:5, 1:10, 1:20 and 1:40 in diluent for the calibration (Figure 4). Samples were measured in a dilution of 1:5 resulting in a total measuring interval of 6.9 - 110 U / mL. PBS was used as diluent for both the calibrator and samples.

[0128] A high serum sample was diluted with heat deactivated serum to cover a major part of the measuring range. The samples were measured in triplicate, and the measured concentration was compared to the theoretical concentration based on the dilution. The results are presented in table 6. The result indicates that the assay is linear between about 15 and 90 U / mL and that samples can be measured with good repeatability240565PC36Table 4: Results from measurement of a serum sample diluted with heat deactivated serum with the turbidimetric analyzer Mindray BS240. The measured concentration is compared to theoretical concentration based on the measured value for 100 % sample and the dilutionHigh Measured CV [%] Theoretical Measured / serum mean n=3 [U / ml] Theoretical [%] [U / ml] [%] 100,0% 88,58 7,6% 88,58 100,0% 66,7% 56,83 1,8% 59,05 96,2% 50,0% 50,28 0,5% 44,29 113,5% 33,3% 31,07 0,6% 29,53 105,2%16,7% 16,39 0,9% 14,76 111,0%Example 10. Assay parameters for the nephelometric analyzer BNII and the 3* FLAG based assay

[0129] The liposome reagent and particle reagent were prepared similarly to the reagent used for the Mindray BS240, except that the activation antibody was formulated as a third reagent. The activation antibody concentration varied between 10 and 100 pg / mL and liposomes of both composition 2, and composition 3 were evaluated. In this specific example liposome composition 3 was used with an activation antibody at a concentration of 100 pg / mL. The assay was programmed as follows:

[0130] Addition of Liposome Reagent (Rl): 150 pL of liposome reagent (Rl) was added to the reaction mixture.

[0131] Addition of sample or calibrator: 8 pL sample or calibrator.

[0132] Incubation: The mixture was incubated at 37 °C for approximately 1 minute.240565PC37

[0133] Addition of activation antibody (100 pg / mL): 4 pL of activation antibody was added.

[0134] Addition of immunoparticles (R.2): 45 pL of immunoparticles was added.

[0135] Reading of the nephelometric signal between 0 and 600 s after addition of the particle reagent.

[0136] The assay was configured as an endpoint reaction, measuring the total change in nephelometric signal from addition of the particle reagent to the end of the reaction (approximately 10 minutes after addition). The selected unit for the assay was U / mL which is the standard unit for liposome-based assays evaluating complement function.Example 11. Calibration and measurement of samples

[0137] The assay using the assay parameters above was calibrated using a single calibrator with the assigned concentration of 55 U / mL. The calibrator was diluted 1:2.5, 1:5, 1:10 and 1:20 in diluent for the calibration (Figure 5). Samples were measured in a dilution of 1:5 resulting in a total measuring interval of 6.9 - 110 U / mL. PBS was used as diluent for both the calibrator and samples.

[0138] A high serum sample was diluted with heat-deactivated serum to cover a significant portion of the measuring range. The samples were measured in triplicate, and the measured concentrations were compared to the theoretical concentrations based on the dilutions. The results, presented in Table 7, indicate that the assay is linear across a significant portion of the desired measuring range and that samples can be measured with good repeatability.240565PC38Table 7: Results from measurement of a serum sample diluted with heat deactivated serum on BNIL The measured concentration is compared to theoretical concentration based on the measured value for 100 % sample and the dilutionHigh Measured CV Theoretical Measured / Theoretical serum mean [%] [U / mL] [%][%] n=3[U / mL]100,0% 67,0 7,9% 67,0 100,0%85,0% 60,0 1,6% 57,0 105,2%60,0% 45,4 4,1% 40,2 112,8%45,0% 35,0 2,4% 30,2 116,1%30,0% 23,5 1,1% 20,1 116,9%Example 12. Investigation of complement-mediated lysis versus molecular weight of released antigen on Mindray BS240

[0139] The assay reagents prepared with 3*-FLAG, 6*FLAG and 3*FLAG conjugates of 4-armed PEG-maleimide (2 kDa or 5 kDa) as described above were analyzed using the Mindray BS240 clinical chemistry analyzer. The liposomes were prepared with an encapsulated concentration of 1.2 mg / mIL for each antigen. The activation antibody was spiked into the detection particles (anti-FLAG coated immunoparticles) at a concentration of 12 pg / mL detection reagent. The assay was performed by adding 100 pL of R1 (liposome reagent) followed by incubation with 15 pL sample that had been manually diluted 1:5 with serum diluent. After approximately 3 minutes of incubation, 50 pL of R2 (anti-FLAG detection particles spiked with anti-DNP antibodies) was added. A blank measurement was taken after 35 seconds, and the reaction was monitored between 3 and 6 minutes at a wavelength of240565PC39546 nm. The instrument parameters used for the assay are summarized in Table 8 below.Table 8. Instrument parameters used in Example 12Parameters Mindray BS-240R.1 volume [pL] 100R.2 volume [pL] 50Sample volume [pL] 15Wavelength [nm] 546Second wavelength NAFirst reading time 10[cycle]Second reading time 34[cycle]Unit U / mLReaction type EndpointCalibration curve splineformulaCalibration level 6

[0140] Calibration of the assay was performed using normal serum with a concentration of 81.3 U / mL. The calibrator was automatically diluted by the instrument using serum diluent to generate calibration levels corresponding to 105.1, 79.9, 60.1, 40.0, 20.1 and 12.5 U / mL. Calibration measurements were performed in duplicate, and the calibration curve was generated using a spline fit.

[0141] Samples were manually diluted 1:5 in serum diluent prior to analysis. In order to compare complement-mediated release with detergent-240565PC40mediated release, 10% deoxycholate in water was used as a reference sample for detergent-mediated lysis.

[0142] All tested FLAG conjugates produced measurable signals in the complement-mediated liposome assay, demonstrating that the conjugated peptides remained functional and detectable after conjugation. The conjugates tested included 3 x FLAG, 6 x FLAG, 2 kDa PEG-MAL-3xFLAG and 5 kDa PEG-MAL-3xFLAG. The apparent molecular weights of the conjugates increased as expected with the addition of PEG. Size exclusion chromatography indicated approximate apparent molecular weights of 6.6 kDa for the 6xFLAG conjugate, 12.3 kDa for the 2 kDa PEG-MAL-3xFLAG conjugate and 18.2 kDa for the 5 kDa PEG-MAL-3xFLAG conjugate.

[0143] Figure 9 is a graph showing the dose response relationships (calibration curves) obtained in Example 12 using liposome reagents containing different conjugates. The general shape of the curves was similar, although differences were observed between the different liposome compositions. The 6xFLAG conjugate displayed a particularly strong signal, and a hook effect was observed for serum concentrations above approximately 80 U / mL.

[0144] To investigate the effect of antigen size on complement-mediated release, the release obtained with complement activation was compared to release induced by 10% deoxycholate. For complement-mediated release, a serum sample with a CH50 level of approximately 50 U / mL was analyzed after dilution 1:5 in serum diluent. Deoxycholate treatment was used to determine the detergent-mediated release of antigen from the liposomes.

[0145] All conjugates generated detectable complement-mediated release, confirming that the different FLAG constructs function in the assay240565PC41system. To evaluate whether molecular size influences the ability of released molecules to pass through complement induced pores formed by the membrane attack complex, the ratio between deoxycholate-induced release and complement-mediated release was calculated for each conjugate. The release ratio was calculated as delta AU for addition of 10 % deoxycholate divided by delta AU for addition of serum at 50 U / mL.

[0146] The results are presented in Figure 10, a bar diagram showing the recovery release (delta AU 10% deoxycholate / delta AU serum 50 U / ml) for 4 different conjugates, 3xFLAG, 6XFLAG, 2k-PEG-3xFLAG, and 5k-PEG-3xFLAG. A high ratio indicates that detergent-mediated release is substantially higher than complement-mediated release, whereas a low ratio indicates relatively efficient release through complement induced pores.

[0147] A clear trend was observed in which increasing conjugate size resulted in reduced relative release through complement induced pores. Smaller peptides such as 3 x FLAG showed lower ratios, whereas larger PEG-conjugated species exhibited higher ratios, indicating that complement-mediated release becomes less efficient as the molecular size increases. These findings suggest that increasing molecular size reduces the efficiency with which released molecules can pass through the complement induced pores, consistent with steric limitations associated with the pore size formed during complement-mediated membrane attack.

[0148] Taken together, the results demonstrate that all investigated 3xFLAG-based conjugates function in the liposome-based CH50 assay and are capable of generating detectable signals following complement-mediated lysis. Increasing the molecular weight of the released antigen affected the efficiency of complement-mediated release, but conjugates within the tested240565PC42size range still produced sufficient signal for turbidimetric or nephelometric detection when combined with the detection particles.

[0149] The influence of molecular size becomes particularly evident when comparing the FLAG conjugates to larger protein antigens such as ovalbumin. Ovalbumin, with a molecular weight of approximately 45 kDa, required encapsulation at a concentration of approximately 50 mg / mL in order to generate a measurable signal in the assay. Such high protein concentrations significantly increase solution viscosity, which in turn results in very high pressures during liposome extrusion. This complicates liposome preparation and may negatively affect process robustness and reproducibility. So, in this context, ovalbumin functions as a negative control, showing that a molecular weight of 45 kDa doesn't work satisfactorily, confirming the upper limit of about 40 kDa.

[0150] In contrast, the FLAG conjugates investigated in this study have apparent molecular weights in the range of approximately 3-20 kDa.Liposomes containing these 4cd conjugates could be prepared using substantially lower encapsulated concentrations, typically in the range of 1-5 mg / mL, while still producing sufficient complement-mediated release to generate a detectable turbidimetric or nephelometric signal in the presence of the detection particles. The lower concentration reduces viscosity and allows liposome extrusion to be performed under significantly milder conditions.

[0151] Although the largest conjugates showed somewhat reduced complement-mediated release relative to detergent-mediated release, the observed signals were still adequate for assay detection. This indicates that antigens in the molecular weight range of approximately 3-20 kDa represent240565PC43a favorable choice, balancing efficient complement-mediated release and practical liposome preparation.

[0152] Taken together, these findings suggest that medium-sized peptide or PEG-peptide conjugates provide clear advantages over larger protein antigens for use in liposome-based CH50 assays. Their smaller size allows efficient release through complement induced pores while enabling liposome preparation at moderate antigen concentrations, thereby avoiding the viscosity and extrusion challenges associated with larger proteins such as ovalbumin. At the same time, these peptides or conjugates offer multiple binding sites (same or different) for the detection antibodies.

[0153] The above results, as well as ongoing experiments, indicate that the inventive liposome-based assay for evaluating complement activity is equally applicable to both turbidimetric and nephelometric clinical chemistry platforms, here represented by the analyzers BS-240 (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) and the BN™ II System (Siemens Healthineers).

[0154] Another advantage is that the herein disclosed assay offers a reliable alternative to the use of mammalian erythrocytes. Further, unlike existing closed and hardware specific systems, the claimed assay can be used on open-channel, fully automated systems already installed at hospitals and clinical laboratories, regardless of these operate according to the turbidimetric or nephelometric assay principle. The claimed assay is directly applicable to operating on existing high-throughput models from manufacturers such as Abbott, Beckman Coulter, Mindray, Roche, and Siemens Healthineers. The claimed assay also works on smaller, benchtop models, such as the Selectra MACH5 from Vital Scientific.240565PC44

[0155] Without further elaboration, it is believed that a person skilled in the art can, using the present description, including the examples, utilize the present invention to its fullest extent. Also, although the invention has been described herein with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.

[0156] Thus, while various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

240565PC45Claims1. An assay method for quantifying complement activity, the method comprising the following steps:mixing a serum sample and a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance,activating the surface-modified liposomes,adding a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles,reacting the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension under conditions allowing complement activation, liposome lysis, release of the first reagent substance, and particle agglutination, measuring the degree of agglutination turbidimetrically or nephelometrically to determine complement activity.

2. The method according to claim 1, wherein the encapsulated first reagent substance is a peptide or polynucleotide having a molecular weight of about 2 to about 40 kDa, preferably in the interval of about 2 to about 30 kDa, and wherein the binding partner specific for the encapsulated first reagent substance is chosen from binding moieties, aptamers, and antibodies exhibiting binding specificity for said encapsulated first reagent substance.

3. The method according to claim 1, wherein the encapsulated first reagent substance is selected from peptides and polynucleotides having a240565PC46molecular weight of about 2 to about 40 kDa, preferably in the interval of about 2 to about 30 kDa; and wherein the binding partner specific for the encapsulated first reagent substance is an antibody or mixture of antibodies selected from monoclonal or polyclonal IgA, IgG, and IgM antibodies binding specifically to the encapsulated first reagent substance.

4. The method according to claim 1, wherein the encapsulated first reagent substance is a multiple, a combination or a conjugate comprising a peptide or peptides chosen from DYKDDDDK-tag, polyglutamate-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag, NE-tag, Avi-tag, PA-tag, His-tag, and E2-tag, said multiple, combination, or conjugate comprising at least 15 amino acids.

5. The method according to claim 1, wherein the surface modification of the surface-modified liposomes comprises the presence of an antigen, an antibody, or a biologically active protein bound to the surface of the liposomes.

6. The method according to claim 1, wherein the surface-modified liposomes are composed of phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and cholesterol.

7. The method according to claim 1, wherein the surface-modified liposomes are phospholipid-based, and wherein the surface modification comprises the presence of an antigen, an antibody, or a biologically active protein covalently bound to phosphatidyl ethanolamine in the phospholipid composition of the liposomes.240565PC478. The method according to claim 7, wherein activation of the surface-modified liposomes is induced by addition of a binding partner with specific affinity for the antigen, antibody, or biologically active protein conjugated to the liposomes.

9. The method according to claim 8, wherein said binding partner is added with the liposome suspension.

10. The method according to claim 8, wherein said binding partner is present in the detection reagent suspension and the activation induced when the detection reagent suspension is added to the mixture of sample and liposome suspension.

11. The method according to claim 1, wherein said binding partner is included in a separate activation reagent added prior to the reaction step.

12. The method according to claim 1, wherein the particles in the detection reagent suspension are coated with a binding partner specific for the encapsulated first reagent substance, and said particles are selected from the group consisting of latex particles, magnetic beads, gold nanoparticles, and microspheres.

13. The method according to claim 1, wherein agglutination is detected by nephelometric or turbidimetric measurement performed at an optical wavelength from about 400 to about 900 nm.

14. An assay kit for determining complement activity, comprising:a liposome reagent suspension comprising surface-modified liposomes encapsulating a first reagent substance,240565PC48a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, a reagent capable of activating the surface-modified liposomes, andone or more buffer solutions for maintaining assay conditions wherein said encapsulated first reagent substance is a peptide or polynucleotide having a molecular weight of about 2 to about 40 kDa, preferably in the interval of about 2 to about 30 kDa, andwherein the binding partner specific for the encapsulated first reagent substance is chosen from binding moieties, aptamers, and antibodies exhibiting binding specificity for said encapsulated first reagent substance.

15. A computer program product comprising computer-executable instructions which, when executed on a processor, cause a clinical analyzer to perform a method according to any one of claim 1 - 13.

16. A computer program product comprising computer-executable instructions which, when executed on a processor, cause a clinical analyzer to perform a method comprising the following steps:mixing a serum sample and a liposome reagent suspension comprising surface-modified liposomes that encapsulate a first reagent substance,activating the surface-modified liposomes,adding a detection reagent suspension comprising particles coated with a binding partner specific for the encapsulated first reagent substance, wherein binding induces agglutination of the particles,240565PC49reacting the serum sample, the liposome reagent suspension containing activated liposomes, and the detection reagent suspension, under conditions allowing complement activation, liposome lysis, release of the first reagent substance, and particle agglutination, and measuring the degree of agglutination turbidimetrically or nephelometrically to determine complement activity,wherein the program is configured to:receive assay data corresponding to the degree of agglutination measured turbidimetrically or nephelometrically,process the assay data to quantify complement activity, and output a quantified complement activity value or a corresponding diagnostic result.

17. The computer program according to claim 15 or 16, wherein the clinical analyzer is an automated clinical analyzer.

18. A method for analysing and evaluating complement activity using machine learning, the method comprising:performing the assay method according to any one of claims 1 to 13 to obtain assay data indicative of complement activity, inputting the assay data into a trained machine learning model configured to correlate assay signals with complement activity levels, processing the assay data using the machine learning model to generate a quantified value or classification of complement activity status, andoutputting the quantified value or classification result.240565PC5019. The method of claim 18, wherein the machine learning model is trained using a dataset comprising historical assay results and corresponding clinical diagnoses.

20. The method of claim 18, wherein the step of processing the assay data using the machine learning model comprises anomaly detection for identifying atypical complement activity profiles.

21. A method for remotely analysing complement activity assay data, the method comprising:receiving assay data generated by the method according to any one of claims 1-13 from a remote laboratory device,transmitting the assay data to a cloud-based server for processing,applying a trained machine learning model to process the assay data and determine a quantified value,transmitting the processed complement activity result back to the remote laboratory device for display or further analysis.

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