Methods and compositions for assaying blood levels of legumain

Abstract

In one aspect, a homogenous assay method for determination of blood levels of legumain molecules (an asparaginyl endopeptidase) is disclosed. In one aspect, the assay utilizes specific sizes of nanoparticles that arc coated with antibody or antibodies specifically towards legumain molecule or its fragment(s). In one aspect, the assay is designed in a homogenous manner with a dynamic range from 0.2 to 160 ng/mL. In another aspect, disclosed herein is a kit for assaying blood levels of legumian comprising two parts of reagents (R1 and R2), which kit is adaptable to be used on clinical chemistry analyzers. In one aspect, the cut-off values for differentiating normal from high risk of cancers such as breast cancer, colorectal cancer and stomach cancer are 18±3 ng/mL.

1 . A single-step or one-step method using heat-stressed magnetic microparticles in a legumain immunoassay for detecting legumain concentration in a blood, serum or plasma sample containing or suspected of containing legumain at concentrations as low as 0.01 ng/ml, comprising:
 (a) contacting the blood, serum or plasma sample with a plurality of magnetic microparticles which are prepared by a method comprising:
 (i) coating each of the plurality of magnetic microparticles with a legumain capturing antibody or an antigen binding molecule that specifically binds the legumain or legumain fragment thereof; 
 (ii) heat-stressing the magnetic microparticles coated with the legumain capturing antibody or the antigen binding molecule thereof for between about 1.0 hour and about 10 days at a temperature of between about 30° C. and about 60° C., 
   wherein the contacting further comprises an addition of a detecting antibody that specifically binds to the legumain molecule at a different binding epitope compared with the legumain capturing antibody or the antigen binding molecule thereof, and the detecting antibody is labeled with chemiluminescent signal generating molecule, and optionally the chemiluminescent signal generating molecule comprises an acridinium ester (AE), an aminobutylethylisolumiol (ABEI), an alkaline phosphatase or a fluorescence generating molecule, wherein optionally the fluorescence generating molecule comprises a fluorescein molecule, and a chemiluminescent signal is generated upon binding of the legumain capturing antibody or the antigen binding molecule thereof to the legumain to form an antibody-legumain conjugate,   (b) detecting the chemiluminescent signal using a photomultiplier, and optionally the antibody-legumain conjugate is washed with a wash buffer before the detecting step ; and   (c) determining the level of legumain or legumain fragment in the blood, serum or plasma sample using a calibrator curve and comparing with a calibrator or calibrated set of a predetermined levels of legumain, where the calibration curve is constructed by using a calibrator or calibrated set of predetermined concentration of legumain.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . A legumain immunoassay kit for determining the level of legumain in a sample, comprising:
 (a) a buffer with its pH value ranging from about 3.0 to about 10.0, or about 5.0 to about 9.0;   (b) a plurality of magnetic microparticles coated with a capturing antibody or an antibody fragment specifically against human legumain or a legumain fragment of a degradation fragment thereof; and   (c) a detecting antibody or an antibody fragment that specifically binds to the legumain molecule labeled with a chemiluminescent signal generating molecule,   and optionally also comprising a calibrator or calibrator set and/or a control or a control set.   
     
     
         5 . The kit of  claim 4 , wherein:
 the magnetic microparticle has a diameter ranging from about 5 nm to about 2000 nm; and optionally the microparticle has a diameter ranging from about 40 nm to about 400 nm; and optionally the microparticle is a magnetic particle.   
     
     
         6 . The method of  claim 1 , where the diameters of magnetic microparticles are between about 0.1 and about 5.0 μm, and preferably between about 0.5 and about 2.0 μm. 
     
     
         7 . The method of  claim 1 , where the sensitivity or detection limit of the magnetic microparticle based immunoassay is as low as about 0.01 ng/ml. 
     
     
         8 . The method of  claim 1 , further comprising a step (d), comprising the level of legumain in the sample with a normal reference range or a cut-off value. 
     
     
         9 . The method of  claim 1 , wherein the legumain if of mammalian origin. 
     
     
         10 . The method of  claim 9 , wherein the legumain is of human origin. 
     
     
         11 . The method of  claim 1 , wherein the legumain is an asparaginyl endopeptidase or the legumain has an asparaginyl endopeptidase activity. 
     
     
         12 . The method of  claim 1 , wherein the antibody or antigen binding molecule thereof is recombinantly produced or is a monoclonal or polyclonal antibody. 
     
     
         13 . The method of  claim 1 , wherein the magnetic microparticle coated with the antibody or antigen binding molecule is heat stressed at 45° C. for about 1 to about 5 days. 
     
     
         14 . The method of  claim 1 , wherein the sample comprises a serum or plasma sample from a human, or a non-human mammal. 
     
     
         15 . The method of  claim 1 , wherein the magnetic microparticle based immunoassay is a single-step assay wherein the sample, the capturing antibody coated magnetic microparticles and the signal generating molecule labelled detecting antibody are mixed and incubated to form sandwich complex in the same step.