Complement assays and uses thereof

Abstract

The present invention provides methods for assessing complement activation and methods for assessing the ability of an agent or condition of interest to modulate complement activation. The present invention provides methods for assessing whether a subject has or is at increased risk of developing a complement-mediated disorder, e.g., age-related macular degeneration (AMD). Also provided are kits containing materials useful for performing the methods.

We claim: 
     
         1 . A method of assessing complement activation in a sample comprising determining the amount of iC3b, the ratio of iC3b to intact C3, or the fraction of C3 that is intact in the sample. 
     
     
         2 . The method of  claim 1 , wherein the method comprises determining the ratio of iC3b to intact C3 in the sample. 
     
     
         3 . The method of  claim 1 , wherein the sample comprises whole blood, plasma, or serum. 
     
     
         4 . The method of  claim 1 , wherein the sample comprises aqueous humor or vitreous humor. 
     
     
         5 . The method of  claim 1 , wherein the sample was obtained from a human subject. 
     
     
         6 . The method of  claim 1 , wherein the sample was obtained from a subject having or suspected of having a complement deficiency or complement-mediated disease or condition. 
     
     
         7 . The method of  claim 1 , wherein the sample was obtained from a subject suffering from AMD. 
     
     
         8 . The method of  claim 1 , wherein the sample was obtained from a subject who has recently suffered severe injury, stroke, asthma exacerbation, cardiac arrest, or heart attack. 
     
     
         9 . The method of  claim 1 , wherein the sample was obtained from a subject who has been treated with a complement inhibitor. 
     
     
         10 . The method of  claim 1 , wherein the sample comprises an agent being assessed for its ability to modulate complement activation. 
     
     
         11 . The method of  claim 1 , comprising steps of:
 (a) determining the concentration of intact C3 present in the sample;   (b) determining the concentration of iC3b present in the sample;   (c) calculating the ratio of iC3b to intact C3 in the sample using the results of steps (a) and (b).   
     
     
         12 . The method of  claim 1 , comprising steps of:
 (a) determining the concentration of intact C3 present in the sample;   (b) determining the concentration of total C3 present in the sample; and   (c) calculating the fraction of C3 that is intact in the sample using the results of steps (a) and (b).   
     
     
         13 . The method of  claim 1 , comprising steps of:
 (a) capturing intact C3 present in one or more sub-samples of the sample using a first capture agent that binds to intact C3;   (b) quantifying intact C3 captured in step (a);   (c) capturing iC3b present in one or more sub-samples of the sample using a second capture agent that binds to a neoepitope of iC3b;   (d) quantifying iC3b captured in step (c); and   (e) calculating the ratio of iC3b to intact C3 in the sample using the results of steps (b) and (d).   
     
     
         14 . The method of  claim 1 , comprising steps of:
 (a) capturing intact C3 present in one or more sub-samples of the sample using a first capture agent that binds to intact C3;   (b) quantifying intact C3 captured in step (a);   (c) capturing total C3 present in one or more sub-samples of the sample using a second capture agent that binds to total C3;   (d) quantifying total C3 captured in step (c); and   (e) calculating the fraction of C3 that is intact in the sample using the results of steps (b) and (d).   
     
     
         15 . The method of  claim 13  or  14 , wherein the first capture agent, the second capture agent, or both, are each immobilized on a support. 
     
     
         16 . The method of  claim 15 , wherein the support comprises a microwell plate. 
     
     
         17 . The method of  claim 15 , wherein the support comprises a plurality of particles. 
     
     
         18 . The method of  claim 13  or  14 , wherein the capture agent of step (a), step (c), or both comprises an antibody. 
     
     
         19 . The method of  claim 13  or  14 , wherein the capture agent of step (a) comprises an antibody that binds to C3a. 
     
     
         20 . The method of  claim 19 , wherein the antibody is monoclonal. 
     
     
         21 . The method of  claim 13 , wherein the capture agent of step (c) comprises an antibody that binds to a neoepitope of iC3b. 
     
     
         22 . The method of  claim 21 , wherein the antibody is monoclonal. 
     
     
         23 . The method of  claim 14 , wherein the capture agent of step (a) comprises an antibody that binds to C3d. 
     
     
         24 . The method of  claim 14 , wherein step (b) comprises detecting intact C3 using a detection agent that specifically binds to C3a. 
     
     
         25 . The method of  claim 13 , wherein step (a) comprises capturing intact C3 using a monoclonal antibody that binds to C3a, and step (b) comprises detecting intact C3 using a polyclonal antibody that binds to C3. 
     
     
         26 . The method of  claim 13 , wherein step (c) comprises capturing iC3b using a monoclonal antibody that binds to a neoepitope of iC3b, and step (b) comprises detecting iC3b using a polyclonal antibody that binds to C3. 
     
     
         27 . The method of  claim 14 , wherein the capture agent that binds to total C3 is a polyclonal antibody that binds to C3d and the specific binding agent for detecting intact C3 is a monoclonal antibody that binds to C3a. 
     
     
         28 . The method of  claim 14 , wherein step (d) comprises detecting total C3 using a detection agent that binds to total C3. 
     
     
         29 . The method of  claim 28 , wherein the detection agent for detecting total C3 comprises a monoclonal antibody that binds to C3d. 
     
     
         30 . The method of  claim 28 , wherein the detection agent for detecting total C3 comprises a polyclonal antibody that binds to C3. 
     
     
         31 . The method of  claim 1 , wherein the method comprises determining the ratio of iC3b to intact C3, further comprising determining the concentration of total C3 in the sample. 
     
     
         32 . A method of assessing complement activation in a subject comprising steps of: (a) providing a biological sample obtained from the subject; and (b) assessing complement activation in the sample according to the method of  claim 1 . 
     
     
         33 . The method of  claim 32 , further comprising (c) using the result of step (b) to provide diagnostic, prognostic, or treatment-related information regarding the subject. 
     
     
         34 . A method of assessing complement activation in a sample comprising (a) capturing and detecting intact C3, wherein intact C3 is captured using an antibody that binds to C3a but does not bind to other C3 fragments; and (b) capturing and detecting iC3b, wherein iC3b is captured using an antibody that binds to a neoepitope of iC3b, and wherein the ratio of iC3b to intact C3 serves as an indicator of the extent of complement activation, with a higher ratio indicating a greater extent of complement activation. 
     
     
         35 . The method of  claim 34 , wherein the antibody that binds to C3a is a monoclonal antibody. 
     
     
         36 . The method of  claim 34 , wherein the antibody that binds to iC3b is a monoclonal antibody. 
     
     
         37 . The method of  claim 34 , wherein intact C3 and iC3b are detected using the same detection agent. 
     
     
         38 . The method of  claim 33 , wherein intact C3 and iC3b are detected using a polyclonal antibody. 
     
     
         39 . The method of  claim 38 , wherein the polyclonal antibody binds to C3. 
     
     
         40 . The method of  claim 34 , wherein intact C3 is captured using an antibody that binds to C3a and is detected using an antibody that binds to C3. 
     
     
         41 . The method of  claim 34 , wherein intact C3 is captured using a monoclonal antibody that binds to C3a and is detected using a polyclonal antibody that binds to C3. 
     
     
         42 . The method of  claim 34 , wherein intact C3 is captured using a monoclonal antibody that binds to C3a and is detected using a polyclonal antibody that binds to C3d. 
     
     
         43 . The method of  claim 34 , wherein iC3b is captured using a monoclonal antibody that binds to a neoepitope of iC3b and is detected using a polyclonal antibody that binds to C3. 
     
     
         44 . The method of  claim 34 , wherein total C3 is captured using a polyclonal antibody that binds to C3d and is detected using a monoclonal antibody that binds to C3d. 
     
     
         45 . The method of  claim 34 , wherein intact C3 is captured using a monoclonal antibody that binds to C3a and is detected using a polyclonal antibody that binds to C3, and iC3b is captured using a monoclonal antibody that binds to a neoepitope of iC3b and is detected using a polyclonal antibody that binds to C3. 
     
     
         46 . The method of  claim 45 , wherein the same polyclonal antibody is used to detect intact C3 and to detect iC3b. 
     
     
         47 . The method of  claim 34 , wherein the antibody of step (a) and the antibody of step (b) are attached to supports. 
     
     
         48 . The method of  claim 34 , wherein the sample comprises serum or plasma. 
     
     
         49 . The method of  claim 34 , wherein the sample comprises aqueous or vitreous humor. 
     
     
         50 . The method of  claim 34 , wherein the sample was obtained from a human subject. 
     
     
         51 . The method of  claim 34 , wherein the sample was obtained from a subject having or suspected of having a complement deficiency or complement-mediated disease or condition. 
     
     
         52 . The method of  claim 34 , wherein the sample was obtained from a subject suffering from AMD. 
     
     
         53 . The method of  claim 34 , wherein the sample was obtained from a subject who has recently suffered severe injury. 
     
     
         54 . The method of  claim 34 , wherein the sample was obtained from a subject being treated with a complement inhibitor. 
     
     
         55 . The method of  claim 34 , wherein the sample comprises an agent being assessed for its ability to modulate complement activation. 
     
     
         56 . A method of assessing complement activation in a subject comprising steps of: (a) providing a sample obtained from the subject; and (b) assessing complement activation in the sample according to the method of  claim 34 . 
     
     
         57 . The method of  claim 56 , further comprising using the result of step (b) to provide diagnostic, prognostic, or treatment-related information regarding the subject. 
     
     
         58 . A kit comprising: (a) a capture agent that binds to intact C3 and does not bind to C3b or iC3b; (b) a capture agent that binds to a neoepitope of iC3b; and (c) a detection agent that binds to intact C3 and (d) a detection agent that binds to iC3b. 
     
     
         59 . The kit of  claim 58 , wherein the capture agent of (a) comprises a monoclonal antibody that binds to C3a. 
     
     
         60 . The kit of  claim 58 , wherein the capture agent of (b) comprises a monoclonal antibody that binds to a neoepitope of iC3b. 
     
     
         61 . The kit of  claim 58 , wherein the detection agent of (c) and the detection agent of (d) are the same. 
     
     
         62 . The kit of  claim 58 , wherein the detection agent that binds to intact C3 comprises an antibody that binds to C3. 
     
     
         63 . The kit of  claim 62 , wherein the antibody that binds to C3 is a polyclonal antibody. 
     
     
         64 . The kit of  claim 58 , wherein the kit comprises (i) a polyclonal antibody that binds to C3; and (ii) a monoclonal antibody that binds to C3a. 
     
     
         65 . The kit of  claim 58 , wherein the kit comprises (i) a monoclonal antibody that binds to a neoepitope of iC3b; and (ii) a monoclonal antibody that binds to C3a. 
     
     
         66 . The kit of  claim 58 , wherein the kit comprises (i) a polyclonal antibody that binds to C3; (ii) a monoclonal antibody that binds to a neoepitope of iC3b; (iii) and a monoclonal antibody that binds to C3a. 
     
     
         67 . The kit of  claim 58 , further comprising at least one item selected from the group consisting of: instructions for use of the kit to assess complement activation; one or more native complement components or cleavage products; human serum complement, optionally characterized for levels of one or more complement components or cleavage products; serum depleted of one or more complement components; a detectably labeled secondary antibody; an enzyme substrate; a buffer; and a support. 
     
     
         68 . The kit of  claim 67 , wherein the kit comprises one or more proteins selected from the group consisting of: C3 protein; C3d polypeptide; C3b polypeptide, iC3b polypeptide. 
     
     
         69 . The kit of  claim 67 , wherein the detectably labeled secondary antibody is labeled with an enzyme. 
     
     
         70 . A method for assessing whether a subject has, or is at increased risk of developing, age-related macular degeneration (AMD), or is at increased risk of progressing from early AMD to more advanced AMD, comprising detecting intact C3 in a sample comprising serum, plasma, or blood from the subject, wherein a level of intact C3 that is greater than a control level indicates that the individual has or is at increased risk of developing AMD, or is at increased risk of progressing from early AMD to more advanced AMD. 
     
     
         71 . The method of  claim 70 , wherein the control level is a level found in a control population composed of individuals not having AMD. 
     
     
         72 . The method of  claim 71 , wherein the control population is composed of individuals at least 60 years of age. 
     
     
         73 . The method of  claim 70 , wherein the method comprises detecting intact C3 using an ELISA assay. 
     
     
         74 . The method of  claim 70 , wherein the method comprises capturing intact C3 using a monoclonal antibody that binds to C3a and detecting captured intact C3 using a polyclonal antibody that binds to C3. 
     
     
         75 . The method of  claim 70 , wherein the method comprises capturing intact C3 using a monoclonal antibody that binds to C3a and detecting intact C3 using a polyclonal antibody that binds to C3d and does not significantly recognize C3 epitopes outside C3d. 
     
     
         76 . The method of  claim 74  or  75 , wherein the monoclonal antibody is mouse anti-human C3a/C3 antibody #HM2075 (Cell Sciences) or an antibody that binds to the same epitope. 
     
     
         77 . The method of  claim 70 , wherein the subject has not been diagnosed with AMD. 
     
     
         78 . The method of  claim 70 , wherein the individual has early AMD, and wherein a level of intact C3 that is greater than a control level indicates that the individual is at increased risk of progressing from early AMD to more advanced AMD. 
     
     
         79 . The method of  claim 70 , wherein the individual has early AMD, and wherein a level of intact C3 that is greater than a control level indicates that the individual is progressing to more advanced AMD. 
     
     
         80 . A method for assessing whether a subject has or is at increased risk of developing age-related macular degeneration (AMD) comprising determining the level of one or more proteins in a sample comprising serum, plasma, or blood from the subject using a monoclonal antibody that binds to C3a/C3, wherein a level of said one or more proteins that is greater than a control level indicates that the individual has or is at increased risk of developing AMD. 
     
     
         81 . The method of  claim 80 , wherein the method comprises capturing said one or more proteins using the monoclonal antibody that binds to C3a and detecting captured intact C3 using a polyclonal antibody that binds to C3. 
     
     
         82 . The method of  claim 80 , wherein the method comprises capturing said one or more proteins using the monoclonal antibody that binds to C3a and detecting captured intact C3 using a polyclonal antibody that binds to C3d and does not significantly recognize C3 epitopes outside C3d. 
     
     
         83 . The method of  claim 80 , wherein the monoclonal antibody is mouse anti-human C3a/C3 antibody #HM2075 (Cell Sciences) or an antibody that binds to the same epitope. 
     
     
         84 . The method of  claim 80 , wherein the control level is a level found in a control population composed of individuals not having AMD. 
     
     
         85 . The method of  claim 84 , wherein the control population is composed of individuals at least 60 years of age. 
     
     
         86 . The method of  claim 80 , wherein the subject has not been diagnosed with AMD. 
     
     
         87 . A kit comprising: (a) a capture agent that binds to intact C3 and does not bind to C3b or iC3b; and (b) a detection agent that binds to intact C3. 
     
     
         88 . The kit of  claim 87 , wherein the capture agent comprises a monoclonal antibody that binds to C3a. 
     
     
         89 . The kit of  claim 87 , wherein the detection agent comprises a polyclonal antibody that binds to C3. 
     
     
         90 . The kit of  claim 87 , further comprising at least one item selected from the group consisting of: instructions for use of the kit to assess levels of intact C3; intact C3 protein; a detectably labeled secondary antibody; an enzyme substrate; a buffer; and a support. 
     
     
         91 . A method for measuring intact C3 in a sample comprising serum, plasma, blood, or another body fluid obtained from a subject, wherein the method comprises capturing intact C3 using a monoclonal antibody that binds to C3a and detecting captured intact C3 using a polyclonal antibody that binds to C3. 
     
     
         92 . The method of  claim 91 , further comprising comparing the level of intact C3 in the sample with a control level. 
     
     
         93 . The method of  claim 91 , wherein the method comprises detecting intact C3 using an ELISA assay. 
     
     
         94 . The method of  claim 91 , wherein the method comprises capturing intact C3 using a monoclonal antibody that binds to C3a and detecting intact C3 using a polyclonal antibody that binds to C3d and does not significantly recognize C3 epitopes outside C3d. 
     
     
         95 . The method of any of  claims 91 - 94 , wherein the monoclonal antibody is mouse anti-human C3a/C3 antibody #HM2075 (Cell Sciences) or an antibody that binds to the same epitope.