# Patent

## Title
Lectin complement pathway assays and related compositions and methods

## Abstract

This invention is related, in part, to assays for analyzing the lectin complement pathway (LCP) as well as to compositions and methods related thereto.

## Background
RELATED APPLICATIONS
This application is a national stage filing under 35 U.S.C §371 of International Application No. PCT/US2007/003644, filed Feb. 9, 2007, now published as WO 2007/095154 A2, which claims priority under 35 U.S.C. §119 from U.S. provisional application Ser. No. 60/771,880, filed Feb. 10, 2006. The entire contents of which is herein incorporated by reference.


GOVERNMENT SUPPORT
Aspects of the invention may have been made using funding from National Institutes of Health Grant number HL52886, HL56086, DE017821 and/or HL79758. Accordingly, the government may have rights in the invention.


FIELD OF THE INVENTION
This invention is related, in part, to assays for analyzing the lectin complement pathway (LCP) as well as to compositions and methods related thereto.
BACKGROUND OF THE INVENTION
Clinically, mannose binding lectin (MBL) moderates disease severity in both its ‘traditional’ and ‘non-traditional’ capacities. In it's ‘traditional’ role, MRL deficiencies have been linked to recurrent respiratory infections (Cedzynski et al., 2004; Gomi et al., 2004; Zhang et al., 2005; Kaur et al., 2006), meningococcal disease (van Emmerik et al., 1994; Bax et al., 1999; Kuipers et al., 2003; Bathum et al., 2006), and P. aeruginosa infection in burn patients (Moller-Kristensen et al., 2006). Alternatively, MBL recognition of self-antigens has been demonstrated in sterile inflammatory processes including systemic lupus erythematosus (Ohlenscfilaeger et al., 2004; Seelen et al., 2005b; Calvo-Alen et al., 2006; Font et al., 2006), thoracoabdominal aortic aneurysm repair (Fiane et al., 2003), IgA nephritis (Endo et al., 1998; Roos et al., 2001; Hisano et al., 2001; Roos et al., 2006), rheumatoid arthritis (Garred et al., 2000; Saevarsdottir et al., 2001; Gupta et al., 2005; Burton and Dwek, 2006), atherosclerosis (Madsen et al., 1998; Hegele et al., 2000; Spence and Norris, 2003; Sjoholm et al., 2006), diabetes (Hansen et al., 2003; Hansen et al., 2004; Bouwman et al., 2005; Hovind et al., 2005), cancer (Baccarelli et al., 2006; Scudiero et al., 2006) and coronary artery disease (Best et al., 2004; Saevarsdottir et al., 2005). The dichotomous nature of this C-type lectin to recognize both foreign- and/or self-antigens under varying conditions is the focus of intense research, as the appropriate balance of functional MBL and LCP activation may predict outcomes in various disease states.
Several assays are available for the quantification of MBL, either functionally (mannan assay) or non-functionally (sandwich ELISA) (Petersen et al., 2001; Thiel et al., 2002; Roos et al., 2003; Takahashi et al., 2005; Seelen et al., 2005a). However, these assays cannot fully investigate the functional status of the early lectin pathway cascade proteins in a single analysis. Further, activation of the alternative complement pathway directly via MBL (e.g., in the absence of MASP-2) can not be assessed by available assays.
SUMMARY OF THE INVENTION
Provided herein are methods for assessing LCP activation by measuring one or more LCP components in a sample, such as a biological sample. Also provided herein are compositions and other methods related thereto.
In one aspect; the method comprises measuring MBL in a biological sample, measuring another LCP component in the biological sample, and assessing LCP activation in the biological sample. In one embodiment, the method further comprises removing non-covalently bound MBL/MASP-2 complexes. In another embodiment, the other LCP component is MASP-2, C3 or C4. In a further embodiment, the method further comprises measuring a further LCP component in the biological sample. In still another embodiment, the further LCP component is MASP-2, C3 or C4. In yet another embodiment, the other LCP component is C4 and the further LCP component is C3. In still a further embodiment, still another LCP component is measured in the biological sample. In one embodiment, the still other LCP component is MASP-2. In one embodiment, MBL, C4 and C3 are measured in the method. In a further, embodiment, MBL MASP-2, C4 and C3 are measured in the method.
In another aspect, a method of assessing LCP activation is provided, comprising contacting a substrate coated with a ligand of MBL with a biological sample, contacting the sample with a detectably labeled agent that specifically binds MBL, determining the amount of MBL present, and assessing LCP activation in the sample with the results. The method, in one embodiment, further comprises removing non-covalently bound MBL/MASP-2 complexes from the sample. The method, in another embodiment, further comprises contacting the sample with a detectably labeled agent that specifically binds C4. The method, in still another embodiment, further comprises contacting the sample with a detectably labeled agent that specifically binds C3. The method, in a further embodiment, comprises determining the amount of C4 and/or C3 present.
In one embodiment, the ligand of MBL is mannan, mannose or GlcNAc. In another embodiment, the agent that specifically binds MBL is an anti-MBL antibody. In a further embodiment, the agent that specifically binds C4 is an anti-C4 antibody. In yet another embodiment, the agent that specifically binds C3 is an anti-C3 antibody.
In another embodiment, the method further comprises contacting the sample with a detectably labeled agent that specifically binds MASP-2, and determining the amount of MASP-2 present. In one embodiment, the agent that specifically binds MASP-2 is an anti-MASP-2 antibody.
In still another aspect, a method for assessing LCP activation is provided wherein the biological sample contacted with the ligand-coated substrate does not contain non-covalently bound MBL/MASP-2 complexes or has had such complexes removed. The method, in one embodiment, further comprises contacting the sample with a detectably labeled agent that specifically binds C4. The method, in still another embodiment, further comprises contacting the sample with a detectably labeled agent that specifically binds C3. The method, in a further embodiment, comprises determining the amounts of C4 and/or C3 present.
In one embodiment, the ligand of MBL is mannan, mannose or GlcNAc. In another embodiment, the agent that specifically binds C4 is an anti-C4 antibody. In yet another embodiment, the agent that specifically binds C3 is an anti-C3 antibody.
In another aspect, a method for assessing LCP activation is provided that comprises a) incubating the biological sample in one or more wells of a multi-well plate, wherein the wells have been coated with a ligand of MBL (e.g., mannan), b) washing the well or wells and incubating with a composition comprising an antibody that binds specifically to MBL, which optionally is detectably labeled, and c) determining the amount of MBL present. In one embodiment, the composition of step b) further comprises an antibody that binds specifically to MASP-2, which optionally is detectably labeled, and step c) further comprises determining the amount of MASP-2 present. In another embodiment, when two antibodies are used, the labels of the antibodies are either the same or distinct/different. In one embodiment, one label is detected at 700 nm and the other at 800 nm.
In another aspect, the method further comprises d) washing the well or wells containing the sample in a buffer that can be used to remove non-covalently bound MBL/MASP-2 complexes. In one embodiment, the method further comprises e) incubating with an antibody that binds specifically to C3, which optionally is bound to a detectable label that is either the same or different from the other label(s), and f) determining the amount of C3 present. In another embodiment, step e) further comprises incubating with an antibody that binds specifically to C4, which is also optionally attached to a detectable label that is either the same or different from the other label(s) and step f) further comprises determining the amount of C4 present. In one embodiment, when the C3 and C4 antibodies are labeled the labels are distinct.
In still another aspect, a method for assessing MASP-2 function is provided. In one aspect, the method comprises measuring C4 in a biological sample, and determining MASP-2 function with the result. The method, in one embodiment, further comprises measuring another LCP component in the biological sample. In another embodiment, the other LCP component is MBL, MASP-2 or C3. In a further embodiment, the method further comprises measuring a further LCP component in the biological sample. In still another embodiment, the further LCP component is MBL, MASP-2 or C3. In still a further embodiment, still another LCP component is measured in the biological sample. In another embodiment, the method further comprises a step of removing non-covalently bound MBL/MASP-2 complexes.
In any of the methods provided herein, the measurement or detection of MBL can be replaced with the measurement or detection of a ficolin (and the ligand of MBL with a ligand of ficolin as needed) or other activator of the LCP (and ligand as appropriate). Therefore, in a further aspect, a method of assessing lectin complement pathway (LCP) activation, comprising measuring a ficolin in a biological sample, and assessing LCP activation in the biological sample is provided. In one embodiment, the method further comprises measuring another LCP component in the biological sample. In another embodiment, the other LCP component is MASP-2, C3 or C4. In still another embodiment, the method further comprises measuring a further LCP component in the biological sample. In still another embodiment, the further LCP component is MASP-2, C3 or C4. In a further embodiment, the other LCP component is C4 and the further LCP component is C3. In yet a further embodiment, still another LCP component is measured in the biological sample. In another embodiment, the still other LCP component is MASP-2. In one embodiment, the ficolin is L-, H- or M-ficolin.
In one embodiment, in methods which include a step of removing non-covalently bound MBL/MASP-2 complexes, the non-covalently bound MB ASP-2 complexes are removed with the use of a buffer that contains a calcium chelator. In another embodiment, the calcium chelator is EDTA or EGTA. In still another embodiment, the buffer further contains a competitive inhibitor of MBL. In one embodiment, the competitive inhibitor is mannose, N-acetylglucosamine (GlcNAc), fucose, glucose or an anti-MBL antibody.
In one embodiment, the biological sample is a serum, plasma or cerebrospinal fluid sample. In another embodiment, when a plasma sample is analyzed, the method can further include the step of coverting the plasma sample to a serum sample. In one embodiment, the step comprises diluting the plasma sample 1:1 with a VBS++ buffer that is supplemented with calcium chloride. In another embodiment, the step can further comprises removing fibrin clots that develop.
In another embodiment, the biological sample is from a subject with or suspected of having a LCP-mediated disease. In another embodiment, the LCP-mediated disease is acute respiratory distress syndrome, arteriosclerosis, arthritis (e.g., rheumatoid arthritis), atherosclerosis, cancer (e.g., breast cancer, colorectal cancer, esophageal squamous cell carcinoma, lung cancer and prostate cancer), cardiopulmonary bypass, cardiovascular disease, chronic angioedema, coronary artery disease, diabetes, infection (e.g., respiratory infection, P. aeruginosa infection, sepsis (e.g., in burn patients)), autoimmune disease, lupus (e.g., SLE), meningococcal disease, myocardial infarction, Neisseria menningitis, nephritis (e.g., IGA nephritis and membranoproliferative glomerulonephritis), neurological disease, neuropathic pain or stroke. In another embodiment, the subject is one that has undergone or is undergoing dialysis. In a further embodiment, the subject is one in which thoracoabdominal aortic aneurysm repair is occurring or has occurred. In still another embodiment, the subject is one that has undergone transplantation. In yet another embodiment, the subject is one in which ischemia and reperfusion has occurred or is occurring. In one embodiment, the ischemia and reperfusion is gastrointestinal ischemia and reperfusion. In a further embodiment, the subject has ischemic heart disease.
The methods, in one embodiment, are performed with the same biological sample or portion thereof. In another embodiment, the methods are performed in the same area of a substrate to which the sample is contacted. In one embodiment, therefore, the methods are performed in the same well of a well plate.
The methods provided, in one embodiment, are performed with a sample size of less than 500 μl. In another embodiment, the sample size is less than 400 μl, 300 μl, 200 μl, 100 μl or 50 μl. In yet another embodiment, the sample size is 1-20 μl. In a further embodiment, the sample size is 15-20 μl, 10-15 μl, 5-10 μl or 1-5 μl.
In another aspect, kits are provided that can be used to carry out the methods of the invention. In one aspect, a kit is provided comprising an agent that specifically binds MBL, a buffer comprising a calcium chelator, an agent that specifically binds another LCP component, and instructions for assessing LCP activation. In one embodiment, the buffer further comprises a competitive inhibitor of MBL. In another embodiment, the other LCP component is MASP-2, C3 or C4. In a further embodiment, the kit further comprises an agent that specifically binds a further LCP component. In one embodiment, the further LCP component is MASP-2, C3 or C4. In another embodiment, the other LCP component is C4 and the further LCP component is C3. In a further embodiment, the agents that specifically bind are antibodies.
For any of the kits provided herein that include an agent that specifically binds MBL, an agent that specifically binds a ficolin (or other activator of LCP) can be substituted in its place. In another aspect, a kit is provided comprising an agent that specifically binds a ficolin, an agent that specifically binds another LCP component, and instructions for assessing LCP activation. In one embodiment, the other LCP component is MASP-2, C3 or C4. In another embodiment, the kit further comprises an agent that specifically binds a further LCP component. In a further embodiment, the further LCP component is MASP-2, C3 or C4. In yet another embodiment, the other LCP component is C4 and the further LCP component is C3. In one embodiment, the agents that specifically bind are antibodies. In another embodiment, the ficolin is an L-, H- or M-ficolin.
In still another aspect, a kit is provided that comprises a buffer comprising a calcium chelator, an agent that specifically binds C4, and instructions for assessing MASP-2 activation. In one embodiment, the buffer further comprises a competitive inhibitor of MBL.
In another embodiment, the kit further comprises an agent that specifically binds another LCP component. In a further embodiment, the other LCP component is MBL, MASP-2 or C3. In one embodiment, the agent that specifically binds is an antibody. In another embodiment, the agents that specifically bind are antibodies.
In still another aspect, buffer compositions that can be used for removing non-covalently bound MBL/MASP-2 complexes are provided. In one aspect, a buffer composition comprising a calcium chelator and a competitive inhibitor of MBL is provided. In one embodiment, the competitive inhibitor of MBL is mannose, N-acetylglucosamine (GlcNAc), fucose, glucose or an anti-MBL antibody. In another embodiment, the competitive inhibitor is mannose. In still another embodiment, the competitive inhibitor is present at a concentration of at least 30 mM. In a further embodiment, the competitive inhibitor is present at a concentration of 30 mM-300 mM. In yet another embodiment, the calcium chelator is. EDTA or EGTA. In still another embodiment, the calcium chelator is present at a concentration of greater than or equal to 10 mM. In yet a further embodiment, the calcium chelator is present at a concentration of 10 mM-100 mM. In another embodiment, the buffer composition has a pH of 7-8. In a further embodiment, the buffer composition has a pH of 7.4-7.8. In one embodiment, the buffer composition comprises 50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 0.05% Tween-20, 100 mM D(+)-mannose and has a pH 7.8.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides an outline of important steps of the LCP.
FIG. 2 demonstrates the deposition of MBL indicated by binding of the IRDye800-labeled anti-MBL mAb (2A9) and visualized on the 800 nm infra-red channel.
FIG. 3 provides results for MBL and MASP-2 deposition. Triplicate values of each serum undiluted sample and dilutions were averaged and expressed as units of Integrated Intensity for MBL (FIG. 3A). MASP-2 in pooled human serum/plasma were screened for using the second infra-red channel available for analysis (700 nm) (FIG. 3B).
FIG. 4 illustrates the deposition of C4 and C3. FIG. 4A shows that C4 is deposited, as detected by IRDye800-labeled anti-C4. Deposition of C3 on mannan plates was detected by IRDye700-labeled anti-C3 (FIG. 4B).
FIG. 5 demonstrates the key components in LCP activation that can be analyzed using LCP FLISA. Oval outlines delineate the endpoints directly analyzed by the assay.
FIG. 6 provide results from an MBL-dependent LCP assay using LICOR technology. Multiple endpoints of LCP activation are concurrently analyzed on the far-infrared channels 700 nm and 800 nm. MBL (FIG. 6A) was analyzed first at 800 nm over a series of serum dilutions. Following a regeneration step (FIG. 6B) which removes non-covalently associated MBL and MASPs, covalently bound C4b and C3b were then analyzed in the same well (FIG. 6C). The left inset (FIG. 6C) represents MBL-dependent C4b analysis (green—800 nm) and MBL-dependent C3b analysis (red—700 nm), respectively. The right upper and lower insets (FIG. 6C) represent C4b and C3b deposition, respectively.
FIG. 7 illustrates quantitation of MBL in the standard PLS. A pooled human serum standard (PLS) was standardized by using MBL-deficient serum reconstituted with a human MBL standard (Staten Serum Institut, Copenhagen, Denmark). Correlation between detected units of integrated intensity (II) at 800 nm and ligand binding was performed using a linear regression model to establish MBL-dependent binding in ng/ml (FIG. 7A). Specificity of functional MBL binding was demonstrated by blocking with mAb 3F8 (FIG. 7B). Using the PLS to establish functional MBL levels (FIG. 7C), triplicate values of each donor (DN 1=donor 1, DN 2=donor 2, DN 3=donor 3, DN 4=donor 4) serum sample diluted in binding buffer are expressed as mean+/−SE.
FIG. 8 demonstrates quantitation of MBL-dependent C4b deposition. A linear correlation between detected units of integrated intensity (II) at 800 nm using known amounts of C4b plated onto 384 well plates was established (FIG. 8A). The amount of MBL-dependent C4b that was bound to the mannan-coated plates following incubation of the PLS was revealed as a log-linear relationship between the integrated intensity of activated PLS deposited C4b and known amounts of C4b (FIG. 8B). Specificity for MBL-dependent C4b deposition was verified by inhibition with mAb 3F8 (FIG. 8C). Using the PLS to establish MBL-dependent C4b levels, triplicate values of each donor (DN 1=donor 1, DN 2=donor 2, DN 3=donor 3, DN 4=donor 4) serum sample diluted in binding buffer are expressed as mean+/−SE of the PLS for C4b (horizontal bars) and MBL (vertical bars).
FIG. 9 illustrates quantitation of MBL-dependent C3b deposition. A linear correlation between detected units of integrated intensity (II) at 700 nm using known amounts of C3b plated onto 384 well plates was established (FIG. 9A). The amount of MBL-dependent C3b that was bound to the mannan-coated plates following incubation of the PLS was revealed as a log-linear relationship between the integrated intensity of activated PLS deposited C3b and known amounts of C3b (FIG. 9B). Specificity for MBL-dependent C3b deposition was verified by inhibition with mAb 3F8 (FIG. 9C). Using the PLS to establish MBL-dependent C3b levels, triplicate values of each donor (DN 1=donor 1, DN 2=donor 2, DN 3=donor 3, DN 4=donor 4) serum sample diluted in binding buffer are expressed as mean+/−SE of the PLS for C3b (horizontal bars) and MBL (vertical bars).
FIG. 10 provides results from an analysis of functional MBL, and MBL-dependent LCP activation, correlated to MBL genotype. Serum samples from donors with the indicated MBL haplotype were evaluated for functional MBL (FIG. 10A), C4b deposition (FIG. 10B) and C3b deposition (FIG. 10C). Differences between medians of haplotype groups for MBL, MBL-dependent C4b, and MBL-dependent C3b are statistically significant; Kruskal-Wallis ANOVA on ranks (p<0.001). There is a direct correlation to functional MBL concentrations with C4b deposition (FIG. 10D) and C3b deposition (FIG. 10E). The MBL-dependent LCP phenotype can be summarized by evaluating MBL binding, MBL-dependent C4b and C3b deposition together (FIG. 10F).
FIG. 11 provides results from an analysis of MBL-dependent AP amplification. MBL-dependent C4b deposition evaluated in 3-25% sera is inhibited by 3F8 and not anti-factor D (FIG. 11A). Alternatively, MBL-dependent AP ‘tick over’ amplification occurs approaching physiologic serum concentration (13 and 25%), as anti-human MBL mAb 3F8 together with anti-human factor D mAb can inhibit C3b deposition (FIG. 11B), but neither of these mAb can completely inhibit independently. Analysis of MBL-dependent AP amplification at 25% serum shows that although MBL-dependent C4b deposition remains compromised in DN1 (MBL-low/deficient) compared to DN 4 (MBL-high) (FIG. 11C), deficiency in MBL-dependent LCP activation can be rescued at the level of C3 convertases (FIG. 11D) when DN 1 is compared with DN 4 at 25% sera concentration. FIGS. 8D and 9D represent the same donors at 3% sera and demonstrated MBL-dependent C4b and C3b deposition, respectively.
Fig. 12 illustrates an example of a kit. Item 10 provides the kit. Item 12 provides a kit structure. Items 14, 16 and 18 each represent a container in the kit.



DETAILED DESCRIPTION OF THE INVENTION
The humoral response to invading pathogens is mediated by a repertoire of innate immune molecules and receptors able to recognize pathogen-associated molecular patterns (PAMPs). Mannose binding lectin and ficolins, initiation molecules of the lectin complement pathway (LCP), are members of a family of acute phase proteins bridging innate and adaptive immunity. It has become clear that early components of the MBL-dependent LCP also recognize self-antigens and/or IgM following oxidative stress. The concentration of human MBL is determined by the MBL2 gene, which has several haplotypes that determine circulating MBL level and function. Further, evidence suggests direct activation of the alternative complement pathway, independent of the serine protease MASP-2, through an MBL-mediated association. Current assays for MRI, and MASP-2 lack the ability to assess all components of the early LCP in a single assay system to the level of C3 cleavage (e.g., the step needed for opsonization of pathogens). Further, the available assays do not assess the functional state of MBL, MASP-2 as well as the C3 convertase. Therefore, assays have been developed such as a low volume, fluorochrome linked immunoassay (FLISA) that quantitatively assess the functional status of MBL, MASP-2 and C3 convertase. The FLISA can be used, for example, as a high-throughput assay to identify potential deficiencies in the MBL-dependent LCP, as well as to identify specific human disease correlations between these components and clinical outcomes.
Provided herein are methods that allow for the assessment of LCP activation in a biological sample. The assays include the measurement of one or more LCP components in a biological sample. A “LCP component” can be any of the components provided in FIGS. 1 and 5. Such components include, for example, MBL, MASP-2, C4, C3, ficolins (such as L-, H- and M-ficolins), etc. The components also include complexes or breakdown products formed at any step of the pathway, such complexes and breakdown products are also illustrated in FIGS. 1 and 5.
In one embodiment, the methods include the steps of measuring MBL or a ficolin (e.g., L-, H- or M-ficolin) in a biological sample as well as measuring at least one other LCP component in the biological sample. The results from the steps can be used to assess LCP activation. The assessment of LCP activation is the determination of whether or not the LCP is active and/or the determination of the level of activation. The assessment of LCP activation can also refer to the function or activation of various LCP components within the pathway. Therefore, the assessment of LCP activation can also include the assessment of the pathway up to C3 cleavage, the assessment of C3 convertase or the function of MASP-2 (such as through the measurement of C4). “Measuring” or “measurement”, when used in regard to an LCP component, is intended to refer to any assessment of the amount of a particular LCP component, in a sample. “Amount” is intended to include a determination of the presence or absence of the LCP component as well as the level of the LCP component in the sample. The “level” is some quantitation of the LCP component in the sample and can be a relative value or an absolute value. Such measuring or measurement can be accomplished with any means known in the art or provided herein. Quantitation can be performed with methods known in the art and those provided herein, such as in the Examples. In one embodiment, quantitation is performed using standard curves for the LCP component(s) detected.
The measurement of a LCP component, such as MBL, can be performed with any of a number of methods well known in the art. For example, the LCP component can be measured using an agent that specifically binds the LCP component. The agent can then be detected. Detection of binding of the agent to the LCP component can be accomplished, for example, by detecting a label bound to the agent or to a molecule that binds thereto. In some embodiments, the agent is an antibody and the antibody is detectably labeled. In this embodiment, it is the label bound to the antibody agent that is detected to measure the LCP component. In other embodiments, the agent is an antibody and a molecule that binds the agent is detectably labeled. The LCP component, in this embodiment is, therefore, measured by detecting the label bound to the molecule that binds the antibody. This molecule can, in some embodiments, also be an antibody.
The other LCP component can be any of the LCP components as described above. In one embodiment, where MBL or a ficolin is measured, the other LCP component can be MASP-2. In another embodiment, the other LCP component can be C3 or C4. In still another embodiment, C3 and C4 can be measured. In a further embodiment, all four of MBL (or a ficolin), MASP-2, C3 and C4 are measured. In still another embodiment, MBL (or a ficolin), C3 and C4 are measured in order to assess LCP activation.
Methods are also provided where the function of MASP-2 is assessed. Such methods can include the step of measuring C4. In some embodiments, the methods can also include the measurement of another LCP component. Such components have been provided elsewhere herein.
The assays can be performed on any substrate. Substrates include well plates, such as 96 or 384 well plates. In some embodiments, the assay is performed in a single area of the substrate, such as a single well of a well plate. In such embodiments, all of the LCP components that are measured are measured with assay steps within the same well. In other embodiments, one or more LCP components are measured in one well and one or more other LCP components are measured in another well. For example, MBL (or a ficolin), or MBL and MASP-2 can be measured with binding agents that specifically bind thereto in one well and other LCP components, such as C3 and/or C4, can be measured in another well. Replicate samples can be measured with other substrates, or in other areas of the same substrate, such as other wells of the same assay plate, as can one or more control samples. In methods where MBL or ficolin is measured, the substrate can be coated with a ligand of MBL or ficolin. Such ligands are known in the art and include MBL ligands such as mannan, mannose or N-acetylglucosamine (GlcNAc). Ligands for ficolin include GlcNAc. Other ligands for MBL and ficolin are known to those of ordinary skill in the art.
The assays, in some embodiments, are performed with the same biological sample or same portion of the biological sample. Alternatively, steps of the assays can be performed with more than one biological sample or more than one portion of a biological sample. The assays, in other embodiments, can be performed with a small amount of sample. In some embodiments, the sample size is less than 500 μl, 400 μl, 300 μl, 200 μl, 100 μl or 50 μl. In still another embodiment, the sample size is 15-100 μl. In yet another embodiment, the sample size is 1-20 μl. In a further embodiment, the sample size is 15-20 μl, 10-15 μl, 5-10 μl or 1-5 μl.
In some embodiments, one or more of the LCP components (such as MBL, ficolin or MASP-2) can be removed prior to performing the rest of the steps of a method for assessing LCP activation. For example, in some embodiments where LCP activation via ficolin measurement is desired, MBL can be removed from the biological sample. In other embodiments, where LCP activation via MBL measurement is desired, ficolins can be removed. Techniques for removing a LCP component are known to those of ordinary skill in the art. For example, centrifugation is used, or antibodies directed to the LCP component are used and the sample processed using chromatography.
Any of the methods provided can further include the step of removing non-covalently bound complexes, such as MBL/MASP-2 complexes, from the sample. Non-covalently bound complexes, such as MBL/MASP-2 complexes, can be removed with the use of a buffer that includes a calcium chelator, such as EDTA or EGTA. Other chelators will be apparent to those of ordinary skill in the art. The chelators can be present, in some embodiments, in the buffer composition at a concentration of greater than or equal to 10 mM. In other embodiments, the chelator is present at a concentration of between 10-100 mM, such as 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM. The buffers can also include a competitive inhibitor, such as a competitive inhibitor of MBL. “Competitive inhibitors of MBL” include mannose, GlcNAc, fucose, glucose or an agent that specifically binds MBL, such as an anti-MBL antibody. Other competitive inhibitors are known to those of ordinary skill in the art. The competitive inhibitor can be present, in some embodiments, at a concentration of at least 30 mM. In other embodiments the competitive inhibitor is present at a concentration of 30-300 mM, such as 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM or 300 mM. The buffer compositions can, in some embodiments, have a pH of 7-8. In other embodiments, the buffer has a pH of 7.4-7.8, such as 7.4, 7.5, 7.6, 7.7 or 7.8. In one embodiment, the buffer contains 50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 0.05% Tween-20, 100 mM D(+)-mannose and has a pH 7.8. Compositions comprising the buffers described herein are also provided.
The assays can, in some embodiments, include washing, mixing and stirring steps as necessary or as otherwise described herein, particularly in the Examples. Including such steps in any of the methods provided herein is well within the routine knowledge of one of ordinary skill in the art.
The assays/methods provided can be used with any of a number of detection systems, in addition to the Odyssey Infrared Imaging System (LICOR) or Aerius detection systems, and one of ordinary skill in the art can make appropriate simple and routine modifications as necessary. Any modifications that may be needed (e.g., with the use of enzyme linked antibodies or other fluorescent antibodies), depending on the detection system used, are well within the ordinary skill of those in the art. Other examples of detection systems include fluorescent plate readers, luminometers, spectrophotometer plate readers, etc. One of ordinary skill in the art will readily understand how to perform the assays/methods provided using any of these detector systems as well as others known in the art. Optionally, the detector systems can be coupled to a liquid handling system and/or an automatic plate washer such that numerous samples can be easily and quickly analyzed. The assays provided herein can be, therefore, performed in a high throughput format.
The measurement of an LCP component (e.g., MBL) can be accomplished using an agent that specifically binds to the LCP component. Agents that specifically bind MBL include those that specifically bind MB; when not complexed to MASP-2 as well as those that specifically bind to MBL when complexed with MASP-2. Agents that specifically bind MASP-2 include those that specifically bind MASP-2 when not complexed to MBL as well as those that specifically bind to MASP-2 when complexed with MBL. Agents that specifically bind to C3 include those that bind C3, C3b or some other cleavage product thereof. Agents that specifically bind to C4 include those that bind C4, C4b or to some other cleavage product thereof. Such agents can also include those that bind a complex that includes C4 or a portion thereof, such as the C4b2a complex.
An “agent that specifically binds” is any molecule that binds with a greater affinity to the molecule of interest than to some other molecule (in some embodiments, an unrelated molecule). Typically, the agent binds with an affinity that is at least two-fold greater than its affinity for binding to other molecules. In some embodiments, the agents can bind with an avidity and/or binding affinity that is 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 70-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold or more than that exhibited by binding to another molecule.
The agents that specifically bind can be binding peptides, such as antibodies or antigen binding fragments thereof. The binding peptides, in some embodiments, can be isolated. As used herein, the term “isolated” means that the agents are substantially pure and are essentially free of other substances with which they may be found, for example, in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, the agents are sufficiently pure and are sufficiently free from other constituents, such as, in the case of binding peptides, biological constituents of their hosts cells so as to be useful in, for example, the assays/methods provided herein. Because an agent may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the agent may comprise only a small percentage by weight of the preparation. The agent is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated, for example, in living systems.
Binding peptides may be synthesized or produced by recombinant means by those of skill in the art. Methods for preparing or identifying peptides which bind to a particular target are well known in the art. Molecular imprinting, for instance, may be used for the de novo construction of macromolecular structures such as peptides which bind to a particular molecule. See, for example, Kenneth J. Shea, Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Klaus Mosbach, Molecular Imprinting, Trends in Biochem. Sci., 19(9) January 1994; and Wulff, G., in Polymeric Reagents and Catalysts (Ford, W. T., Ed.) ACS Symposium Series No. 308, pp 1.86-230, American Chemical Society (1986). One method for preparing mimics of binding peptides involves the steps of: (i) polymerization of functional monomers around a known binding peptide or the binding region of an antibody (the template) that exhibits a desired activity; (ii) removal of the template molecule; and then (iii) polymerization of a second class of monomers in the void left by the template, to provide a new molecule which exhibits one or more desired properties which are similar to that of the template. In addition to preparing peptides in this manner other binding molecules, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials can also be prepared. This method is useful for designing a wide variety of biological mimics that are more stable than their natural counterparts, because they are typically prepared by the free radical polymerization of functional monomers, resulting in a compound with a nonbiodegradable backbone. Other methods for designing such molecules include, for example, drug design based on structure activity relationships which require the synthesis and evaluation of a number of compounds and molecular modeling.
Peptides which bind to a LCP component may also be identified by conventional screening methods such as phage display procedures (e.g., methods described in Hart, et al., J. Biol. Chem. 269:12468 (1994)). Hart et al. report a filamentous phage display library for identifying novel peptide ligands for mammalian cell receptors. In general, phage display libraries using, e.g., M13 or fd phage, are prepared using conventional procedures such as those described in the foregoing reference. The libraries display inserts containing from 4 to 80 amino acid residues. The inserts optionally represent a completely degenerate or a biased array of peptides. Ligands that bind are obtained by selecting those phages which express on their surface a ligand that binds to the molecule of interest. These phages then are subjected to several cycles of reselection to identify the peptide ligand-expressing phages that have the most useful binding characteristics. Typically, phages that exhibit the best binding characteristics (e.g., highest affinity) are further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the phage surface and the optimum length of the expressed peptide to achieve optimum binding. Alternatively, such peptide ligands can be selected from combinatorial libraries of peptides containing one or more amino acids. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counterparts.
To determine whether a peptide binds to a LCP component any known binding assay may be employed. For example, the peptide may be immobilized on a surface and then contacted with a labeled LCP component. The amount of the LCP component which interacts with the peptide or the amount which does not bind to the peptide may then be quantitated to determine whether the peptide binds to the LCP component A surface, for example, having a monoclonal antibody with known specificity immobilized thereto may serve as a positive control.
Screening of binding peptides, also can be carried out utilizing a competition assay. If the peptide being tested competes with, for example, a monoclonal antibody with known specificity or some other ligand, as shown by a decrease in binding of the monoclonal antibody or ligand, then it is likely that the peptide and the monoclonal antibody or ligand bind to the same, or a closely related, epitope. Still another way to determine whether a peptide has the specificity of a monoclonal antibody or ligand with known specificity is to pre-incubate the monoclonal antibody (or ligand) with the LCP component with which it is normally reactive, and then add the peptide being tested to determine if the peptide being tested is inhibited in its ability to bind the LCP component. Other assays will be apparent to those of skill in the art.
As mentioned above, the binding peptide can be an antibody or fragment thereof. Antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term “antibody” means not only intact antibody molecules but also fragments of antibody molecules retaining binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. In particular, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab′)2, and Fab. F(ab′)2, and Fab fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
According to one embodiment, the antibody is an intact soluble monoclonal antibody. An intact soluble monoclonal antibody, as is well known in the art, is an assembly (f polypeptide chains linked by disulfide bridges. Two principle polypeptide chains, referred to as the light chain and heavy chain, make up all major structural classes (isotypes) of antibody. Both heavy chains and light chains are further divided into subregions referred to as variable regions and constant regions. As used herein the term “monoclonal antibody” refers to a homogenous population of immunoglobulins which specifically bind to an epitope (i.e. antigenic determinant).
Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding-of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology. Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions of the antibody, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc′ region has been enzymatically cleaved, or which has been produced without the pFc′ region, designated an F(ab′)2 fragment, retains both of the antigen binding sites of an intact antibody. An isolated F(ab′)2 fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd (heavy chain variable region). The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
The terms Fab, Fc, pFc′, F(ab′)2 and Fv are used consistently with their standard immunological meanings [Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford)].
Therefore, antibodies of the invention may be single chain antibodies or may be single domain antibodies (intrabodies or intracellular antibodies). Intrabodies are generally known in the art as single chain Fv fragments with domains of the immunoglobulin heavy (VH) and light chains (VL). Well-known functionally active antibody fragments include but are not limited to F(ab′)2, Fab, Fv and Fd fragments of antibodies. These fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). For example, single-chain antibodies can be constructed in accordance with the methods described in U.S. Pat. No. 4,946,778 to Ladner et al. Such single-chain antibodies include the variable regions of the light and heavy chains joined by a flexible linker moiety. Methods for obtaining a single domain antibody (“Fd”) which comprises an isolated variable heavy chain single domain, also have been reported (see, for example, Ward et al., Nature 341:644-646 (1989), disclosing a method of screening to identify an antibody heavy chain variable region (VH single domain antibody) with sufficient affinity for its target epitope to bind thereto in isolated form). Methods for making recombinant Fv fragments based on known antibody heavy chain and light chain variable region sequences are known in the art and have been described, e.g., Moore et al., U.S. Pat. No. 4,462,334. Other references describing the use and generation of antibody fragments include e.g., Fab fragments (Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevieer, Amsterdam, 1985)), Fv fragments (Hochman et al., Biochemistry 12: 1130 (1973); Sharon et al., Biochemistry 15: 1591 (1976); Ehrilch et al., U.S. Pat. No. 4,355,023) and portions of antibody molecules (Audilore-Hargreaves, U.S. Pat. No. 4,470,925). Thus, those skilled in the art may construct antibody fragments from various portions of intact antibodies without destroying the specificity of the antibodies for their target.
As is well-known in the art, the complementarity determining regions (CDRs) of an antibody are the portions of the antibody which are largely responsible for antibody specificity. The CDRs directly interact with the epitope of the antigen. In both the heavy chain and the light chain variable regions of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The framework regions (FRs) maintain the tertiary structure of the paratope, which is the portion of the antibody which is involved in the interaction with the antigen. The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3 contribute to antibody specificity. Because these CDR regions and in particular the CDR3 region confer antigen specificity on the antibody these regions may be incorporated into other antibodies or peptides to confer the identical specificity onto that antibody or molecule.
Antibodies to the LCP components can be produced with methods routine in the art. The following is a description of a method for developing a monoclonal antibody specific for MBL. The description is exemplary and is provided for illustrative purposes only.
Female Balb/C mice are initially inoculated (i.p.) with 250 ul of the following mixture: 250 μl Titermax mixed with 100 μg human MBL in 250 μl PBS. The following week and for three consecutive weeks the mice are injected with 50 μg hMBL in 250 PBS. On the 4th week the mice are injected with 25 μg MBL in 250 μl PBS and the mice are fused 4 days later. The fusion protocol is adapted from Current Protocols in Immunology. The splenocytes are fused 1:1 with myelinoma fusion partner P301 from ATCC using PEG 150 at 50% w/v. The fused cells are plated at a density of 1.25×106/m with 100 μl/well of a 96 well microtiter plate. The fusion media consists of Deficient DME high glucose, Pen/Strep (50,000 U pen, 50,000 μg strep per liter), 4 mM L-glutamine, 20% fetal bovine serum, 10% thyroid enriched media, 1% OPI, 1% NEAA, 1% HAT, and 50 μM 2-mercaptoethanol. The cells are fed 100 μu/well on day one and 100/well media are exchanged on days 2, 3, 4, 7, 9, 11, and 13. The last media change before primary screening consists of HAT substituted for the 1% HT. All subsequent feedings are done with fusion media minus the minus HT or HAT. Screening is done with human MBL plated to plastic FLISA plates (96 well plates). Purified hMBL is plated in each well at 50 μl volume containing 2 μg/ml MBL in 2% sodium carbonate buffer. The plates are then blocked with 3% BSA in PBS. Tissue culture media (50 μl) is placed in the wells and incubated for 1 hour at room temperature. The plates are washed and a secondary HRP labeled goat anti-mouse IgG antibody is used for detection. Colorimetric analysis is done with ABTS and read at 405 nm. Positive controls consists of a polyclonal antibody to human MBL. Cells are then grown in media consisting of the following: DMEM high glucose no-I-glut, sod, pyruvate 500 ml (Irvine Scientific #9024), heat inactivated Hyclone 10%, 1% Non-essential amino acids, 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. All positive wells are then screened for function in a secondary screen.
The antibodies may be human antibodies (e.g., a human monoclonal antibody) or intact humanized monoclonal antibodies. A “humanized monoclonal antibody” as used herein is a monoclonal antibody or functionally active fragment thereof having human constant regions and an antigen-binding region (e.g., CDR3) from a mammal of a species other than a human. Humanized monoclonal antibodies may be made by any method known in the art. Humanized monoclonal antibodies, for example, may be constructed by replacing the non-CDR regions of a non-human mammalian antibody with similar regions of human antibodies while retaining the epitopic specificity of the original antibody. For example, non-human CDRs and optionally some of the framework regions may be covalently joined to human FR and/or Fc/pFc′ regions to produce a functional antibody. There are entities in the United States which will synthesize humanized antibodies from specific murine antibody regions commercially, such as Protein Design Labs (Mountain View Calif.). For instance, a humanized form of a murine anti-MBL antibody could be prepared and used according to the methods of the invention.
European Patent Application 0239400, the entire contents of which is hereby incorporated by reference, provides an exemplary teaching of the production and use of humanized monoclonal antibodies in which at least the CDR portion of a murine (or other non-human mammal) antibody is included in the humanized antibody. Briefly, the following methods are useful, as examples for constructing a humanized monoclonal antibody including at least a portion of a mouse CDR. A first replicable expression vector including a suitable promoter operably linked to a DNA sequence encoding a variable domain of an immunoglobulin (Ig) heavy or light chain and the variable domain comprising framework regions from an human antibody and a CDR region of a murine antibody is prepared. Optionally a second replicable expression vector is prepared which includes a suitable promoter operably linked to a DNA sequence encoding at least the variable domain of a complementary human Ig light or heavy chain respectively. A cell line is then transformed with the vector(s). Preferably the cell line is an immortalized mammalian cell line of lymphoid origin, such as a myeloma, hybridoma, trioma, or quadroma cell line, or is a normal lymphoid cell which has been immortalized by transformation with a virus. The transformed cell line is then cultured under conditions known to those of skill in the art to produce the humanized antibody.
As set forth in European Patent Application 0239400 several techniques are well known in the art for creating the particular antibody domains to be inserted into the replicable vector. (Vectors and recombinant techniques are discussed in greater detail below.) For example, the DNA sequence encoding the domain may be prepared by oligonucleotide synthesis. Alternatively a synthetic gene lacking the CDR regions in which four framework regions are fused together with suitable restriction sites at the junctions, such that double stranded synthetic or restricted subcloned CDR cassettes with sticky ends could be ligated at the junctions of the framework regions. Another method involves the preparation of the DNA sequence encoding the variable CDR containing domain by oligonucleotide site-directed mutagenesis. Each of these methods is well known in the art. Therefore, those skilled in the art may construct humanized antibodies containing a murine CDR region without destroying the specificity of the antibody for its epitope.
Human monoclonal antibodies may be made by any of the methods known in the art, such as those disclosed in U.S. Pat. No. 5,567,610, issued to Borrebaeck et al., U.S. Pat. No. 5,565,354, issued to Ostberg, U.S. Pat. No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133: 3001 (1984), Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, p. 51-63 (Marcel Dekker, Inc, new York, 1987), and Boerner et al., J. Immunol., 147: 86-95 (1991). In addition to the conventional methods for preparing human monoclonal antibodies, such antibodies may also be prepared by immunizing transgenic animals that are capable of producing human antibodies (e.g., Jakobovits et al., PNAS USA, 90: 2551 (1993), Jakobovits et al., Nature, 362: 255-258 (1993), Bruggermann et al., Year in Immuno., 7:33 (1993) and U.S. Pat. No. 5,569,825 issued to Lonberg).
Alternatively, monoclonal antibodies to the various LCP components known in the art may be used in the assays provided herein. For example, known antibodies that bind MBL include monoclonal antibodies produced by the hybridomas deposited with the ATCC under ATCC Accession No. (HB-12621), ATCC Accession No. (HB-12620) and ATCC Accession No. (HB-12619); anti-C4 antibodies can be obtained from Cappel/ICN Pharmaceuticals (Aurora, Ohio), and anti-C3 antibody (mouse anti-human C3 clone 7C12) is known in the art (Tosic et al., 1989).
The agents that specifically bind to the LCP component(s) can be detectably labeled. Detectable labels include near-infrared fluorochromes, radiolabels, fluorescent labels, chromophores, enzyme labels, free radical labels, avidin-biotin labels or bacteriophage labels. Conjugation of the agent and label can be accomplished using techniques known to the art, such as methods of protein conjugation or dye labeling as performed by Rockland Immunochemicals (Gilbertsville, Pa.) and the methods of Chard, Laboratory Techniques in Biology, “An Introduction to Radioimmunoassay and Related Techniques,” North Holland Publishing Company (1978).
Near-infrared fluorochromes include IRDye® 800CW, IRDye® 700DX and AlexaFluor 680.
Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorescamine.
Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, and the oxalate esters.
Typical bioluminescent compounds include luciferin, and luciferase. Typical enzymes include alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, glucose oxidase, and peroxidase.
The labels when more than one LCP component is measured in an assay can be the same or they can be different. For example, labels that can be detected at different wavelengths can be used. When different labels are used, in some embodiments, the labels can be individually detected and quantitated. Alternatively, the different labels can be detected at different points in time when the other label is no longer present. As provided above, near-infrared fluorochromes can be used and include those that can be detected at 700 nm and those that can be detected at 800 nm.
“Biological samples”, as used herein, are any samples from a subject in which the assessment of LCP activation can be made. Such samples include, for example, serum, plasma and cerebrospinal fluid samples. In some embodiments, where plasma samples are analyzed; the methods can include a step whereby the plasma sample is converted to a serum sample. Methods for doing so can, in some embodiments, include using a VBS++ buffer supplemented with calcium chloride (such as with a 1:1 dilution of sample to buffer). The methods can further include removing the fibrin clots that result.
The samples can be obtained, in some embodiments, from subjects that have or are suspected of having a LCP-mediated disease. Such subjects are readily identifiable by those of ordinary skill in the art. A “LCP-mediated disease” as used herein is any condition, disease or disorder which involves in its onset or pathogenesis of LCP complement activation. Therefore, included are those that involve cellular injury caused by LCP complement activation. LCP-mediated diseases include, for example, acute respiratory distress syndrome, arteriosclerosis, arthritis (such as rheumatoid arthritis), atherosclerosis, cancer (such as breast cancer, colorectal cancer, esophageal squamous cell carcinoma, lung cancer and prostate-cancer), cardiopulmonary bypass, cardiovascular disease, chronic angioedema, coronary artery disease, diabetes, dialysis, infection (such as respiratory infection, P. aeruginosa infection, sepsis (such as in burn patients)), ischemia and reperfusion (such as that involved in ischemic heart disease and gastrointestinal ischemia and reperfusion), autoimmune disease, lupus (such as SLE), meningococcal disease, myocardial infarction, Neisseria menningitis, nephritis (such as IGA nephritis and membranoproliferative glomerulonephritis), neurological disease, neuropathic pain, stroke, thoracoabdominal aortic aneurysm repair and transplantation. Each of these is well-known in the art and/or described, for instance, in Harrison's Principles of Internal Medicine (McGraw Hill, Inc., New York).
The methods provided herein can be used to assess LCP activation in a subject having or suspected of having a LCP-mediated disease. Such methods can also include the step of selecting a treatment for the subject based on the LCP activation assessment. The treatment selected can be based on the particular type of LCP-mediated disease the subject has and/or the results from the assays provided herein.
The methods provided can also be used to compare samples from subjects that have or are suspected of having a LCP-mediated disease with samples from subjects that are disease free or from the general population.
The methods provided can also be used to examine samples from subjects that have suffered from a myocardial infarction or have undergone cardiovascular surgery.
Kits are also provided that can be used for performing the methods of the invention provided herein. In one embodiment, therefore, a kit is provided that includes an agent that specifically binds MBL and an agent that specifically binds at least one other LCP component. The kit, therefore, can include an agent that specifically binds MBL and an agent that specifically binds MASP-2. In another embodiment, the kit can include an agent that specifically binds MBL and an agent that specifically binds C3 or C4. In still another embodiment, the kit includes an agent that specifically binds MBL, an agent that specifically binds MASP-2, an agent that specifically binds C3 and an agent that specifically binds C4. In yet another embodiment, the kit includes an agent that specifically binds MBL, an agent that specifically binds C3 and an agent that specifically binds-C4.
In another embodiment, a kit is provided that includes an agent that specifically binds ficolin and an agent that specifically binds at least one other LCP component. The kit, therefore, can include an agent that specifically binds ficolin and an agent that specifically binds MASP-2. In another embodiment, the kit can include an agent that specifically binds ficolin and an agent that specifically binds C3 or C4. In still another embodiment, the kit includes an agent that specifically binds ficolin, an agent that specifically binds MASP-2, an agent that specifically binds C3 and an agent that specifically binds C4. In yet another embodiment, the kit includes an agent that specifically binds ficolin, an agent that specifically binds C3 and an agent that specifically binds C4.
In still another embodiment, a kit is provided that includes an agent that specifically binds C4.
The kits provided can also include a buffer comprising a calcium chelator. The buffer, in some embodiments, can also include a competitive inhibitor, such as a competitive inhibitor of MBL. Such inhibitors are described elsewhere herein.
The kits provided can also include instructions for assessing LCP activation and/or MASP-2 function.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLES
Example 1
Measurement of MBL, MASP-2, C4 and C3
Materials and Methods
Plate Preparation.
384-well clear flat bottom HB microplates (Corning Incorporated, Corning, N.Y.) were coated with 25 μI per well of 0.5 mg/ml mannan (Sigma, St. Louis, Mo.) in sodium carbonate/bicarbonate buffer, pH 9.8. Plates were incubated overnight at 40° C. The following day, plates were blocked with 110 μl per well of 3% BSA in PBS without calcium or magnesium (Sigma) for two hours at room temperature. Plates were then washed three times with PBS/Tween 20 (0.5%), once with PBS, and once with veronal buffered saline (VBS). Excess volume was removed, and plates were stored at −30° C.
IRDye-Labeled Antibodies
All antibodies were labeled using long-wavelength near-infrared fluorochromes (Rockland Immunochemicals, Gilbertsville, Pa.) IRDye800 or IRDye700. Fluorochrome to protein quality control was performed by Rockland Immunochemicals, and optimal dilutions for ELISA analysis were defined. Antibodies used for conjugation were as follows: IRDye800 labeled 2A9 (mouse anti-recombinant human MBL), IRDye800 goat anti-human C4 (ICN Pharmaceuticals, Aurora, Ohio), IRDye700 mouse anti-recombinant human MASP-2 and IRDye700 goat anti-human C3 (ICN Pharmaceuticals).
MBL-Dependent LCP ELISA
All ELISA analyses were performed using the Odyssey Infrared Imaging system (LICOR Biosciences, Lincoln, Nebr.), and all sample processing for plates utilized a fully automated liquid handling and plate washing system. Prepared 384-well mannan plates were thawed at room temperature (25° C.) for 15 minutes. Pooled human serum (Fisher-MP Biomedicals, Pittsburgh, Pa.) was then added to plates at 15 μI per well and serially diluted using the Precision 96/384 well Microplate Pipetting System (BIO-TEK Instruments Inc., Winooski, Vt.). Sample diluent was hypertonic buffer (20 mM Tris, 1M NaCl, 10 mM CaCl2, 0.05% Triton X, 1% BSA), and hypertonic buffer alone was added to mannan wells as a background control. Plates were then incubated at 37° C. for 30 minutes and then washed (Buffer A: 145 mM NaCl, 10 mM HEPES, 10 mM CaCl2, 490 mM MgCl2, 0.05% Tween) four times using the EIx405 Microplate Washer with Bio-Stack Microplate Stacker (BIO-TEK). MBL and MASP-2 were detected by addition of 15 μl per well of 1:1000 IRDye800 2A9 (anti-MBL) and 1:500 IRDye700 anti-MASP-2 diluted in Wash 10+0.05% Tween, incubated for one hour at 25° C., washed (×3 with Buffer A) and then read on the Odyssey Infrared Imaging system (LICOR) concurrently at 700 nm and 800 nm. Following this first reading, plates were washed three times with Buffer B: (50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 0.05% Tween-20, 100 mM mannose at pH 7.8). Plates were read again on the Odyssey Infrared Imaging system (LICOR)-concurrently at 700 nm and 800 nm to establish baseline fluorescence following removal of the primary antibody cocktail mix. Next, the second antibody cocktail to determine C3 and C4 deposition was performed by addition of 15 μl per well of 1:1000 IRDye700 anti-C3 and IRDye800 anti-C4 (diluted in Buffer A), incubated for one hr at 25° C., washed (4× with Buffer A) and read on the Odyssey Infrared Imaging system (LICOR), concurrently at 700 nm and 800 nm. Analytical accuracy of MBL, MASP-2, C4 and C3 is represented by the average integrated intensities of pooled human serum standards (Fisher-MP Biomedicals) in triplicate per plate, and in triplicate per assay (intra and inter CV values expressed as a percentage). Pooled human serum was assigned a lot number, and frozen in aliquots at −80° C.
Results
Important steps of the LCP are outlined in FIG. 1, which includes the four endpoints of this ELISA: MBL, MASP-2, C4 and C3. Mannan is one of the major oligosaccharide ligands for MBL, and recognition of mannan is indicative of functional MBL activity. Therefore, MBL deposition on mannan-coated substrates is an indicator of functional MBL levels in patient serum/plasma. Deposition of MBL was indicated by binding of the IRDye800-labeled anti-MBL mAb (2A9) and visualized on the 800 nm infra-red channel; a representative example of which can be observed in FIG. 2. Triplicate values of each serum undiluted sample and dilutions were averaged and expressed as units of Integrated Intensity (FIG. 3a).
The mannose binding lectin associated serine protease 2 (MASP-2) is a regulator in the activation of the LCP via its recognition of MBL. MASP-2 activation is required for enzymatic cleavage of C4 and C2 to form the C3 convertase C4b2a, which results in C3 cleavage and deposition. The dual channel technology of the LCP assay was used to analyze levels of MASP-2 present in patient serum/plasma. As observed in FIG. 3b, this enzymatic protease in pooled human serum/plasma can be screened for using the second infra-red channel available for analysis (700 nm).
The next step of this assay regenerates the LCP analysis-platform by removing non-covalently bound MBL and MASP-2 (and their associated antibodies). The regeneration buffer removes the Ca2+ required for the interaction of MBL, MASP-2 and target ligand, while additionally containing a competitive MBL ligand, mannose. Following regeneration, fluorescence on both the 700 and 800 nm channels were returned to background levels (FIG. 3). Because components of LCP activation downstream of MBL and MASP-2 (i.e., C3 and C4) are deposited and covalently associated with the target (mannan plate), regeneration of the analysis platform increases the analysis from a two end-point assay to a four-endpoint analysis of LCP activation.
As indicated in FIG. 1, C4 is an important indicator of MBL ligand-recognition and of functional MASP-2 concentrations. Following the regeneration step, C4 cleavage and deposition on mannan plates incubated with human serum was investigated. The same wells demonstrated successful binding of MBL and MASP-2 on mannan prior to the regeneration step, and background levels of fluorescence after the regeneration step. As seen in FIG. 4a, C4 is deposited on the plate, as detected by IRDye800-labeled anti-C4.
Additionally, using the 700 nm channel activation of the LCP at the level of C3 convertases can be assessed. Deposition of C3 on the plate signifies formation of a functional C3 convertase and thus, C2's presence indirectly. Deposition of C3 on mannan plates was detected by IRDye700-labeled anti-C3 (FIG. 4b).
References for Example 1


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Example 2
Measurement of MBL, C4 and C3
Materials and Methods
Patient Samples and Standard Serum Components
Serum samples were collected from informed consent donors, following IRB approval, from the Brigham and Women's Hospital population, Boston, Mass. Individual serum samples were pooled to create a standardized human sera stock (PLS) for assay evaluation and validation. Each PLS batch was assigned a lot number, and frozen in aliquots at −80° C. for future FLISA use. Serum samples representing various homozygotic MBL haplotypes were from informed consent donors from the Brigham and Women's Hospital, and the Texas Heart Institute, Baylor College of Medicine, Houston, Tex.
The MBL content of the PLS was standardized using MBL-deficient serum and a quality controlled purified MBL protein standard (Staten Serum Institut, Copenhagen, Denmark). Purified complement components C4b and C3b (Complement Technologies Inc., San Diego, Calif.) were used as quality control samples and to standardize the downstream activated components of the MBL-dependent LCP in the PLS.
Antibodies
All labeled antibodies utilized long-wavelength near-infrared fluorochromes IRDye® 800CW or IRDye® 700DX (Rockland Immunochemicals, Gilbertsville, Pa.) or AlexaFluor 680 (Molecular Probes/Invitrogen, Carlsbad, Calif.). Fluorochrome to protein conjugation was performed by Rockland Immunochemicals, or according to manufacturer's instructions for dye labeling. Optimal dilutions for FLISA labeled antibodies were defined by our laboratory in pilot studies. Antibodies used for conjugation are as follows: IRDye8.00 labeled 2A9 (mouse anti-human MBL (Collard et al., 2000), goat anti-human C4 (Cappel/ICN Pharmaceuticals, Aurora, Ohio); AlexaFluor 680 mouse anti-human C3 clone 7C12 (Tosic et al., 1989), and donkey anti-goat IgG IRDye800 (Cappel/ICN)). Optimal concentrations of each antibody were determined in pilot studies.
Plate Preparation
High binding, 384-well, clear, flat bottom microplates (Corning Incorporated, Corning, N.Y.) were coated with 25 μl per well of 0.5 mg/ml mannan (Sigma, St. Louis, Mo.) in sodium carbonate/bicarbonate buffer pH 9.8. Plates were incubated overnight at 4° C. The plates were then blocked with 110 μl per well of 3% BSA in PBS without calcium or magnesium (Sigma) for two hours at room temperature. Plates were then washed three times with PBS/Tween 20 (0.5%), once with PBS, and once with veronal buffered saline (VBS). Excess volume was removed from the plates, which were covered and stored at −30° C.
MBL-Dependent LCP FLISA
All FLISA analyses were performed using the Odyssey and/or Aerius Infrared Imaging systems (LICOR), and all sample processing for plates utilized a fully automated liquid handling and plate washing system (BIO-TEK Instruments Inc., Winooski, Vt.). Prepared 384-well mannan plates (protocol above) were thawed at room temperature (25° C.) for 15 minutes. Diluted plasma (see preparation below) and/or serum were then added to plates at 15 μl per well and serial diluted using the Precision 96/384 well Microplate Pipetting System (BIO-TEK Instruments Inc.). Sample diluent for serum or plasma samples was fortified veronal buffered saline (VBS++; 143 mM NaCl, 5 mM Barbital Sodium Salt, 5 mM MgCl2, 5 mM CaCl2). Buffers alone were added to mannan wells as a background control. Plates were incubated at 37° C. for 30 minutes and then washed with Buffer A: (145 mM NaCl, 10 mM HEPES, 10 mM CaCl2, 490 μM MgCl2, 0.05% Tween) four times (Elx405 Microplate Washer with Bio-Stack Microplate Stacker; BIO-TEK Instruments Inc.). MBL was detected by addition of 15 μl per well of 1:500 IRDye800 2A9 (anti-MBL) diluted in Buffer A incubated for one hour at 25° C., washed (3× with Buffer A) and then read on the Odyssey/Aerius Infrared Imaging system (LICOR) at 800 nm. Following this first reading, plates were soaked for 15 minutes with 100 μl/well of Buffer B: (50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 0.05% Tween-20, 100 mM D(+)-mannose at pH 7.8), followed by 1× wash with Buffer B. Plates were read again on the Aerius/Odyssey Infrared Imaging system (LICOR) at 800 nm to establish baseline fluorochrome emittance following removal of MBL, and the primary antibody against MBL. Next, 15 μl per well of goat anti-human C4 at 1:500 (diluted in Buffer A) was added to plates and incubated for one hour at 25° C. Following a 4× wash with Buffer A; 15 μl per well of 1:1000 IRDye800 donkey anti-goat IgG, and 1:3000 AlexaFluor 680 7C12 (diluted in Buffer A; anti-C3b) were incubated for one hour at 25° C., washed (4× with Buffer A) and read on the Aerius/Odyssey Infrared Imaging system (LICOR) concurrently at 100 nm and 800 nm. Measurement of MBL (ng/ml) was interpolated from the serum or plasma dilution series, and MBL-dependent activation products (pg) were evaluated at 3% serum unless otherwise stated.
For inhibition studies, mAb 3F8 specific for human MBL (Collard et al., 2000) and anti-human factor D antibody (Stahl et al., 2003) at the concentrations indicated were added to serum at 25° C. for 30 minutes prior to plate incubation.
Plasma Preparation
Sera and citrated (3.2%) plasma samples were obtained from the same three donors. Citrated plasma (100 μl) was mixed 1:1 with VBS++ buffer supplemented to 16.3 mM calcium chloride to induce coagulation as described (Clinical Chemistry 48: 255-260, 2002;). Sera and coagulated plasma samples were allowed to clot overnight at 4° C. Sera were separated from fibrin clots by centrifugation and then used in the FLISA.
Freeze-Thaw Effects
Sera samples were aliquoted and subjected to multiple rounds of freezing (−80° C.) and thawing to room temperature. Freeze/thaw samples were then subjected to FLISA analysis.
Statistics
Samples were averaged in triplicate and expressed as mean+/− the standard error (SE). PLS integrated intensities were averaged in triplicate per plate and in quadruplicate per assay with intra- and inter-CV values expressed as a percentage. A power analysis was performed to select the appropriate sample of representative haplotypes to achieve statistical significance for functional MBL evaluation. Significance was defined as p<0.05.
Results
The LCP, including the MBL-dependent LCP is outlined in FIG. 5. As mannan is a major ligand for MBL, evaluation of MBL binding on mannan-coated plates can demonstrate functional MBL levels in a sera or plasma sample. MBL-binding was detected using a directly labeled IRDye800 anti-MBL mAb (2A9) visualized on the 800 nm infra-red channel observed in FIG. 6A. A pooled human serum standard (PLS) was standardized by using MBL-deficient serum (Staten Serum Institut; MBL B/B also recognized as O/O) reconstituted with a known purified human MBL-standard (Staten Serum Institut). Intra-assay coefficient variation was 4%. Correlation between detected units of integrated intensity (II) at 800 nm and ligand binding was performed using a linear regression model to establish MBL-dependent binding in ng/ml (FIG. 7A). Specificity of functional MBL binding was demonstrated by blocking with mAb 3F8 (FIG. 7B) or D-mannose. Using the PLS to establish functional MBL levels, triplicate values of individual donor serum sample diluted in binding buffer were averaged and expressed as ng/ml (FIG. 7C, DN 1-4).
Following analysis of the functional MBL concentrations present, the plate was then regenerated by removing non-covalently bound MBL/MASP-2 complexes (and their associated detector antibodies). The regeneration buffer removes the Ca2+ required for MBL complex binding and competitively inhibits MBL binding by addition of D-mannose to the regeneration buffer. Following regeneration, infrared emittance on the 800 nm channel was returned to background levels (FIG. 6B). Because activated components of LCP activation downstream of MBL/MASP-2 (i.e., C3b and C4b) are deposited and covalently bound, regeneration of the analysis platform increases the analysis from a single end-point assay to a 3-endpoint analysis of MBL-dependent LCP activation from one sera sample.
Enzymatic cleavage of C4, with consequent C4b deposition onto the mannan plate demonstrates functional MASP-2 activity (FIG. 5). Following the regeneration step, MBL/MASP-2 complex dependent C4b and C3b deposition on mannan plates from human serum were analyzed at 800 nm and 700 nm, respectively, after incubation with the detection antibodies described in Materials and Methods. As shown in FIG. 6C (left side panel), C4b and C3b were simultaneously recorded from each well. Individual scans for C4b (green) and C3b (red) are shown in the upper and lower right panels of FIG. 6C, respectively.
A linear correlation between detected units of integrated intensity (II) at 800 nm using known amounts of C4b plated onto 384 well plates is shown in FIG. 8A. Using the data obtained from FIG. 8A, the amount of MBL-dependent C4b that was bound to the mannan coated plates was then determined following incubation of the PLS. As shown in FIG. 8B a log-linear relationship between the integrated intensity of activated PLS deposited C4b and known amounts of C4b was observed. These data were used to standardize the PLS and its ability to deposit known amounts of C4b onto mannan-coated plates. Specificity for MBL-dependent C4b deposition was further verified by the ability of mAb 3F8 to dose-dependently inhibit C4b deposition (FIG. 8C). Serum samples prepared in the previous step for MBL analysis (FIG. 7C, DN1-4) were further evaluated in the same well for MBL-dependent C4b deposition against the PLS using a log-linear regression fit and expressed as MBL-dependent C4b in pg (FIG. 8D).
The classical/lectin C3 convertase, C4b2a, is formed by MBL/MASP-2 cleavage of complement protein C2, concurrent with C4 cleavage and deposition of C4b (FIG. 5). Formation of the C3 convertase demonstrates the capacity of the MBL-dependent LCP to activate the complement cascade once MBL binds to its target ligand. Thus, C3b deposition signifies formation of a functional C3 convertase and as such indirectly allows for evaluation of functional. C2 in a sera/plasma sample (FIG. 5). The dual channel technology of this LCP assay allows for the multiplicity of endpoint evaluation using the 700 nm channel to assess MBL-dependent C3 convertase activity by observation of C3b deposition onto the mannan coated plate.
Similar to the methods used for the standardization for C4b, known amounts of C3b were plated onto 384 well plates. A linear correlation between detected units of integrated intensity (II) at 700 nm using known amounts of C3b plated to 384 well plates is shown in FIG. 9A. Using the data obtained the amount of MBL-dependent C3b that was bound to the mannan coated plates was then determined following-incubation of the PLS. As shown in FIG. 9B a log-linear relationship between the integrated intensity of activated PLS deposited C3b and known amounts of C3b were observed. These data were used to standardize the PLS and its ability to deposit-known amounts of, C3b onto mannan-coated plates. Intra-assay-coefficient variation was 10%. Specificity for MBL-dependent C3b deposition was further verified by the ability of mAb 3F8 to dose-dependently inhibit C3b deposition (FIG. 9C). Serum samples prepared in the previous step for MBL analysis (FIG. 3C, DN 1-4) and C4b analysis (FIG. 8D) were further evaluated in the same well for MBL-dependent C3b deposition against the PLS using a log-linear regression fit and expressed as MBL-dependent C3b in pg (FIG. 9D).
To confirm the precision of this MBL-dependent LCP assay, donors of known MBL haplotype (Table. 1) representing a broad range of functional MBL expression were analyzed for MBL and MBL-dependent C4b and C3b deposition. As observed in FIG. 10A measurement of functional MBL confirms the previously reported levels of functional MBL observed in these haplotypes (Steffensen et al., 2000; Terai et al., 2003). Expanding upon these past observations, the MBL-dependent LCP activation and subsequent C4b (FIG. 10B) and C3b (FIG. 10C) deposition associated with these known MBL-haplotypes are quantified using this novel assay system. Activation of the LCP by the functional MBL complex (e.g., functional MASP-2) reveals a quantifiable direct-proportional increase of functional MBL binding and C4b deposition (FIG. 10D). The capacity for these various MBL haplotypes to also deposit C3b is shown. A quantifiable proportional increase of functional MBL binding and C3b deposition was observed (FIG. 10E). The MBL-dependent LCP phenotype can be summarized by evaluating MBL binding, MBL-dependent C4b and C3b deposition together (FIG. 10F). This 3D graph demonstrates that functional MBL, dictated by individual MBL haplotypes, appears to drive the activation and deposition of effector LCP components.







TABLE 1







Represented MBL Haplotypes











Haplotype
Previously Reported
MBL-LCP Assay




(Homozygotes)
MBL (ng/ml)*
MBL (ng/ml)
n
SE















HYPA/HYPA
2003
(n = 54)
2028
18
364


HYPD/HYPD
61
(n = 1)
2
9
1


LXPA/LXPA
118
(n = 4)
292
12
104


LYPA/LYPA
1738
(n = 2)
2053
4
461


LYPB/LYPB
22
(n = 2)
13
12
5


LYQA/LYQA
1899
(n = 5)
1739
18
383











LYQC/LYQC
0
0
3
0





*from R. Steffensen et al., Journal of Immunological Methods 241 (2000) 33-42 and Terai et., al Eur. J. Imm. 33 (2003) 2755-2763






A proposed interaction of MBL with the alternative pathway (AP) has been evaluated (Selander-et al., 2006). Further, the AP mediated amplification of optimal MBL-dependent LCP-opsinophagocytosis has been demonstrated (Brouwer et al., 2006). C4b and C3b deposition in this assay at 3-25% sera concentrations was evaluated (FIG. 11). As shown in FIG. 11A, C4b deposition is entirely dependent on MBL, as inhibition of MBL with mAb 3F8, but not inhibition of factor D, completely inhibits C4b deposition at all the sera-concentrations evaluated. In contrast, C3b deposition (FIG. 11B) at 13% and 25% sera is dependent on the interactions of MBL and the alternative pathway, as inhibition of MBL (3F8) and factor D (anti-fD) completely inhibited C3b deposition, whereas inhibition of each of these two molecules individually was ineffective. At sera concentrations of 3% and 6%, inhibition of MBL, but not factor D effectively inhibited C3b deposition. Interestingly, in an MBL-deficient (low) donor (DN1), it is clear that MBL-dependent LCP activation (e.g., C4b and C3b deposition) is compromised in comparison to MBL-normal or high expressers (FIG. 8D and FIG. 9D). However, evaluating this same donor in the window of MBL-dependent AP amplification; it is clear that C4b deposition remains relatively compromised (FIG. 8D vs. FIG. 11C) yet C3b deposition recovers to MBL-normal levels (FIG. 9D vs. FIG. 11D). Thus, this assay addresses a potential role of the MBL-dependent pathway to interact with the alternative pathway to increase C3b deposition in MBL-deficient or MBL-low expresser patients.
Plasma samples were also taken from the same individuals (n=3) and subjected to the FLISA as described. While there was a tendency to have higher values for each of the measured parameters, no significant difference was observed (mean±SEM). These data (Table 2) demonstrate that plasma (citrate and likely EDTA or heparin containing clinical sample tubes) can also be used to assay the LCP after recalcification.







TABLE 2







Comparison of plasma and sera samples











MBL (ng/ml)
C4b (pg)
C3b (pg)
















Sera
1505 ± 418
2797 ± 1417
10302 ± 4351



Plasma
2732 ± 576
3552 ± 619
21325 ± 6791










Sera samples were collected from four individuals and run in the FLISA as fresh samples or frozen to −80° C. and thawed to room temperature five times and then run again in the FLISA. Data (Table 3) are means ±SEM for n=4 determinations. No significant differences were observed for MBL, C4b or C3b at any of the individual points. These data demonstrate that frozen samples can be used from established databases or frozen and analyzed at a later date without significant loss information obtained in the frozen/thawed samples.







TABLE 3







Freeze/thaw data












Fresh
1
2
3
4
5










MBL(ng/ml)












1418 ± 314
1344 ± 333
1239 ± 269
1635 ± 436
1431 ± 395
1424 ± 265







C4b (pg)












3118 ± 935
2469 ± 911
2337 ± 776
1562 ± 547
 2713 ± 1102
 2828 ± 1031







C3b(pg)












33800 ± 2551
32836 ± 2844
34424 ± 3556
34408 ± 3601
34379 ± 3300
32906 ± 3326









Discussion

Described herein are assays that include assays that detect the functional status of the LCP utilizing a high-throughput liquid handling system, together with infra-red technology, examples of which are described in the foregoing example and about which the following discussion pertains.
This is the first multi-tiered LCP assay prototype that can successfully analyze the key components of early LCP activation from a single sample. This analysis can be performed for both serum samples and plasma samples. Importantly, the prototype for this multi-endpoint LCP assay demonstrates the shortcomings of currently available assays for analyzing MBL-dependent LCP activation in patient samples, while highlighting the ability of this platform to overcome these challenges. Dual channel detection (FIG. 6C), together with the application of a regeneration buffer (FIG. 6B), allows re-establishment of an evaluation platform to analyze comprehensively the functional capabilities of an individual's LCP. In addition, evaluation can be performed in a single, small volume sample of serum or plasma, making the assay quick and cost efficient. A complete LCP pathway evaluation has yet to be demonstrated in currently available assays, which require additional sample and evaluation of additional endpoints (C4 or C3) in separate experimental steps (Roos et al., 2003), or additional sample and reconstitution with exogenous C4 (Petersen et al.; 2000; Petersen et al., 2001). Evaluation of the LCP in these previously published assays does not represent the physiological contributions of the patient's own C4, including interaction with MASP-2 (Rossi et al.; 2001; Rossi et al., 2005), formation of the C3 convertase, and subsequent C3 activation and deposition.
The example assay reports functional MBL, MBL-dependent C4b, and MBL-dependent C3b activation and deposition in plasma or serum using a pooled serum standard (PLS). The PLS was batched, aliquoted and assigned a lot designation thereby establishing intra-assay consistency as reported in Materials and Methods. A common PLS for multiple endpoint analysis adds to the already efficient and cost-effective analysis platform by essentially combining three individual protein standards into one. The PLS is specific for MBL (FIG. 7), MBL-dependent C4 activation (FIG. 8), and formation of MBL-dependent C3 convertase (FIG. 9), as the anti-MBL mAb 3F8 can abrogate MBL binding and the subsequent C4b and C3b deposition. The PLS was also evaluated against a standard serum control commonly used in other MBL assay protocols, and independently confirmed the functional MBL content as 1 μg/ml.
It is clear that MBL is important clinically as lower levels of MBL predispose certain patients to recurrent infection (Zhang et al., 2005; Bathum et al., 2006), while progressively higher levels of MBL have been associated with post-surgical complications including ischemia/reperfusion (i/R) injury, coronary diseases and more recently, diabetes (Fiane et al., 2003; Best et al., 2004; Saevarsdottir et al., 2005; Bouwman et al., 2005). Functional MBL levels are dictated by a group of SNPs; the most highly represented haplotypes associated with the promoter, the structural gene segment, and the untranslated region affecting both overall production (Madsen et al., 1995) and/or stability and function of the circulating MBL multimer (Dean et al., 2006). Available assay techniques have correlated functional MBL in ng/ml to patient haplotype, therefore a representative panel of MBL haplotypes (Table 1) was selected and evaluated for functional MBL in confirmation of the described MBL-dependent LCP FLISA (FIG. 10A). Evaluated MBL levels were within the standard error of the means (SE) of the previously reported values as indicated in Table 1.
Importantly, the assay is able to take MBL-dependent evaluation further than previously possible, establishing an MBL-dependent LCP activity phenotype including circulating functional MBL, and downstream MBL-dependent LCP activation. For example, multiple MASP-2 SNPs have been identified, and associated with disease prevalence including colorectal cancer and esophageal squamous cell carcinoma (Verma et al., 2006; Sjoholm et al., 2006). Circulating levels of MASP-2 have been evaluated using a sandwich ELISA, although functional levels of MASP-2 in patient samples have yet to be evaluated (Moller-Kristensen et al., 2003). The example MBL-dependent LCP FLISA evaluates functional MASP-2 activity via C4b deposition, as the MBL/MASP-2 complex is required for LCP complement cascade activation through enzymatic cleavage of C4. Recent analysis of MASP-2 SNPs confirmed that functional MASP-2 is required for activation and deposition of C4b, yet the level of C4-activation and C4b deposition may be driven more by functional MBL and not variant MASP-2 (Teillet et al., 2005). As seen in FIG. 8D and FIG. 11D, there is a direct correlation with C4b deposition and circulating functional MBL. The MBL dependent deposition of C4b has been demonstrated previously (Petersen et al., 2001), and although, associated with MBL haplotype (Roos et al., 2003), the magnitude of MBL-dependent activation was not quantified. A quantifiable evaluation of MBL/MASP-2 activation is shown herein.
There is the possibility that C4b deposition in the FLISA could be limited by a hereditary C4 deficiency, and not functional MASP-2-capacity. Complete deficiency of C4 is well documented, although rare, and linked to SLE, membranoproliferative glomerulonephritis, and chronic angioedema (Fishelson et al., 2001; Azofra and Lopez-Trascasa, 2001; Suzuki et al., 2003; Yang et al., 2004; Lhotta et al.; 2004; Sjoholm et al., 2006). Addition of exogenous C4 to a C4b-deposition negative patient sample (Petersen et al., 2000; Petersen et al., 2001), and performing a second evaluation for MBL/MASP-2 dependent C4b deposition would address this issue. Alternatively, functional and quantitative MASP-2 levels may be evaluated independently as a fourth endpoint using an anti-MASP-2 mAb.
The enzymatic activation of C4 by the MBL/MASP-2 initiation complex leads to formation of the classical C3 convertase. Together with C2, C4b2a cleaves C3 into the fluid phase C3a anaphylotoxin and the covalently bound opsonin, C3b (Walport, 2001a; Walport, 2001b). Formation of the C3 convertase is important to the propagation of the LCP enzymatic cascade resulting in clearance of infectious pathogens via opsinophagocytosis, and lytic clearance via the terminal complement complex (TCC, C5b-9). In sterile inflammation, formation of the C3 convertase and C3 cleavage is associated with complement-dependent ischemia and reperfusion injury (Weisman et al., 1990; Souza et al., 2005) and cardiopulmonary bypass (De Silva et al., 2006), IgA nephropathy (Gherghiceanu et al., 2005) and allograft survival (Brown et al., 2006). Interestingly, it has been suggested that restenosis following endarterectomy correlates with high functional MBL (MBL2 haplotype A/A individuals), and increased circulating-C3 (Szeplaki et al., 2006). Evaluation of comprehensive MBL-dependent complement activation at the level of C3 demonstrates a direct correlation of circulating MBL and C3b deposition (FIG. 9D). Furthermore, MBL-dependent C3b deposition proportionally correlates to functional MBL haplotypes (FIG. 10C) further describing what was defined above as an MBL-dependent activation phenotype. It remains to be seen whether this MBL/LCP phenotype is driven by other components of the MBL-dependent LCP in addition to MBL. However, as with activation and deposition of C4, there seems to be a direct correlation of C3 activation and deposition with circulating MBL (FIG. 10E). As described earlier for the MBL/MASP-2 complex activation capacity at the level of C4, MBL-dependent C3 convertase formation is quantifiable.
Similar to C4, there is the possibility that C3b deposition is limited by a hereditary C3 deficiency, or potentially C2 deficiency. The incidence of C3 deficiency is unclear, although clinically, primary C3 deficient patients present with severe infections, and often in early childhood (Reis et al., 2006a). Deficiencies in C3 have also been linked to SLE and membranoproliferative glomerulonephritis (Borzy et al., 1988; Da Silva et al., 2002; Reis et al., 2006b). Alternatively, C2 deficiency is a very common inherited complement deficiency, with an incidence reported at 1:20,000 (Sjoholm et al., 2006). Patients deficient in C2 can be asymptomatic, but may present with SLID or discoid lupus, and increased systemic infections with encapsulated bacteria (Sjoholm et al., 2006). Augmenting a MBL-dependent C3b-deposition negative sample with C3, would likely address which deficiency (i.e., C2 or C3) was involved. In the case of C2 deficiency, addition of C3 to the patient sample would remain C3b-deposition negative as the convertase is not able to form. However, in a C3 deficient patient, addition of C3 to the sample would restore the substrate to the functionally active convertase, resulting in C3b deposition.
Other assay platforms evaluating MBL-dependent C3 activation have looked at MBL-dependent AP amplification or a ‘bypass’ pathway of MBL/C3 interaction (Selander et al., 2006; Brouwer et al., 2006), although MBL-dependent C3 activation was not quantitated in either assay. In addition to the direct evaluation of MBL-specific LCP activation, the FLISA can address the mechanisms of alternate MBL-dependent complement activation proposed (Selander et al., 2006; Brouwer et al., 2006). It is clear that direct MBL-dependent C4b2a activation of the LCP is driven by functional MBL as shown in FIGS. 10 and 11, and as published (Selander et al.; 2006). However, as shown in FIG. 11D, the capacity for MBL-dependent AP amplification may drive the potential for downstream complement activation at the level of C3 convertases with respect to MBL deficient or ‘low’ expressers. The definition of MBL deficiency in the literature is controversial, as some report MBL-deficiency as <50 ng/ml-(Christiansen et al., 1999), <100 ng/ml (Gadjeva et al., 2004) or <500 ng/ml (Ytting et al., 2005; Frederiksen et al., 2006) functional MBL protein. However, one group used <1000 ng/ml as their plateau for correlative MBL deficiency (Neth et al., 2001). With the potential to form even small amounts of C3 convertase, ‘tick-over’ amplification may salvage otherwise limited MBL-dependent LCP capacity during various immunologic challenges. Similarly, a ‘bypass’ mechanism may achieve the same end via different means (Selander et al., 2006). The distinction between the two surrogate MBL-dependent activation routes may be driven by the components of the complement component arsenal unique to each sample (i.e., the relatively common occurrence of C2 deficiency). Furthermore, differential MBL-ligand recognition may play a role in surrogate MBL-dependent complement activation, as a recent evaluation of MBL-dependent/C2-independent C3 activation showed a preference for O-linked mannose in serogroup C(CO) of Salmonella over other mannose-containing serotypes DO and BO (Selander et al., 2006).
An additional member of the lectin family are the ficolins, which recognize and activate the LCP through shared MASPs (Matsushita et al., 2000) as shown in FIG. 5. As it is becoming increasing clear that ficolins may play an important role in human disease, evaluation of ficolin-dependent LCP activation is necessary. Incidence of ficolin deficiency in the general population remains unclear, as does the potential for self-antigen recognition by ficolins. A recent study of gastrointestinal I/R and secondary lung injury showed neutrophil infiltration and C3 deposition independent of MBL or C1q yet dependent on C2 (Hart et al., 2005). As primary injury in this model was MBL-dependent, this lead to the speculation that ficolins played a role in mediating auto-epitope recognition and secondary I/R damage. The versatility of this assay platform allows for the modification of the protocol and evaluation of the contribution of the ficolin-LCP using an alternate functional target (GlcNAc) and antibodies directed towards L-, H- and/or M-ficolins.
The MBL-dependent LCP FLISA extends what is currently understood about functional MBL by describing quantitatively the MBL-dependent LCP activation to the level of C3 convertases. Furthermore, it has been demonstrated that MBL-dependent LCP activation is influenced by the MBL genotype. Because the MBL haplotype alters LCP activation through cleavage and deposition of C3b, an analysis of MBL-dependent complement activation in a MBL-dependent LCP activation phenotype in a single assay is described. This analysis includes the evaluation of MBL-dependent AP amplification, which may play a role in otherwise low MBL expresser individuals. This mechanism of MBL-dependent salvage should be considered with respect to the selective pressure that retaining a high frequency of MBL2 mutation in the population. MBL-deficient/low patients may be protected when it comes to self-antigen recognition following sterile challenge (i.e., I/R injury), yet MBL-dependent AP amplification may impart protection to MBL-deficient individuals by ‘alternatively’ activating the LCP following pathogenic challenge. Utilization of a MBL-dependent LCP. FLISA to evaluate clinical samples collected from donors in differing compromised populations will further the understanding of LCP activation in both infectious and sterile inflammation and injury.
References for Example 2


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The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.
The listing of references herein is not intended to be an admission that any of the references is a prior art reference.

## Claims

1. A method of assessing lectin complement pathway (LCP) activation, comprising:
determining an amount of mannose binding lectin (MBL) in a biological sample,
removing non-covalently bound MBL/mannose binding lectin serine protease 2 (MASP-2) complexes,
determining an amount of another LCP component in the biological sample, and
assessing LCP activation in the biological sample based on the amounts of MBL and the another component.

2. The method of claim 1, wherein the another LCP component is, C3, C3b, C4, C4b or C4b/2a.

3. The method of claim 2, wherein the method further comprises:
measuring a further LCP component in the biological sample.

4. The method of claim 3, wherein the further LCP component is MASP-2, C3, C3b, C4, C4b or C4b/2a.

5. The method of claim 4, wherein the another LCP component is C4 and the further LCP component is C3.

6. The method of claim 4, wherein still another LCP component is measured in the biological sample.

7. The method of claim 6, wherein the still other LCP component is MASP-2.

8. The method of claim 1, wherein the non-covalently bound MBL/MASP-2complexes are removed with the addition of a buffer that contains a calcium chelator.

9. The method of claim 8, wherein the calcium chelator is EDTA or EGTA.

10. The method of claim 8, wherein the buffer further contains a competitive inhibitor of MBL.

11. The method of claim 10, wherein the competitive inhibitor is mannose, N-acetylglucosamine (GlcNAc), fucose, glucose or an anti-MBL antibody.

12. The method of claim 1, wherein the biological sample is a serum, plasma or cerebrospinal fluid sample.

13. The method of claim 1, wherein the biological sample is from a subject with or suspected of having LCP-mediated disease.

14. The method of claim 1, wherein the measurements are performed in the same biological sample or portion thereof.

15. A method of assessing LCP activation, comprising:
contacting a substrate coated with a ligand of MBL with a biological sample,
contacting the sample with a detectably labeled agent that specifically binds MBL,
determining the amount of MBL present,
removing non-covalently bound MBL/MASP-2 complexes from the sample,
contacting the sample with a detectably labeled agent that specifically binds C4,
contacting the sample with a detectably labeled agent that specifically binds C3,
determining the amount of C4 and C3 present, and
assessing LCP activation in the sample based on the amounts of MBL, C4 and C3.

16. The method of claim 15, wherein the ligand of MBL is mannan, mannose or GlcNAc.

17. The method of claim 15, wherein the agent that specifically binds MBL is an anti-MBL antibody.

18. The method of claim 15, wherein the agent that specifically binds C4 is an anti-C4 antibody.

19. The method of claim 15, wherein the agent that specifically binds C3 is an anti-C3 antibody.

20. The method of claim 15, wherein the method further comprises:
contacting the sample with a detectably labeled agent that specifically binds MASP-2, and determining the amount of MASP-2 present.

21. The method of claim 20, wherein the agent that specifically binds MASP-2 is an anti-MASP-2 antibody.

22. The method of claim 15, wherein the biological sample is a serum, plasma or cerebrospinal fluid sample.

23. The method of claim 15, wherein the biological sample is from a subject with or suspected of having a LCP-mediated disease.

24. The method of claim 15, wherein the measurements are performed in the same well of a well plate.