Quantitative assay for heat shock protein 70 (hsp70) protein in body fluids

Abstract

Provided is a novel kit and assay for free and lipid-bound (exosomal) Hsp70. In particular, an ELISA is described for determining the level of Hsp70 in sample derived from a body fluid of a subject, characterized in that the level of Hsp70 is determined by an anti-Hsp70 antibody.

1 . An in vitro method for assaying free and lipid-bound (exosomal) heat shock protein 70 (Hsp70) in a sample derived from a body fluid of a subject, characterized in that the level of Hsp70 is determined by an anti-Hsp70 antibody which is antibody cmHsp70.1 as produced by hybridoma cmHsp70.1 (Accession Number DSM ACC2629), or cmHsp70.2 as produced by hybridoma cmHsp70.2 (Accession Number DSM ACC2630), or a HSP70-binding fragment or biotechnological or synthetic derivative thereof. 
     
     
         2 . The method of  claim 1 , wherein the body fluid is blood and the sample comprises serum or plasma, or urine, preferably wherein the serum is separated from blood by centrifugation after allowing to clot. 
     
     
         3 . The method of  claim 1 , wherein an elevated level of Hsp70 compared to a control sample derived from a healthy subject is indicative for a tumor or an inflammatory disease, preferably multiple sclerosis (MS). 
     
     
         4 . The method of  claim 1 , further comprising the steps of
 (i) diagnosing a tumor in a subject or whether a cancer patient is amenable to the treatment with an anti-tumor agent, in particular Hsp70 specific agent, respectively, wherein an elevated level of Hsp70 compared to a control sample is indicative for the tumor and possibility for the treatment with the agent, respectively; or   (ii) monitoring the treatment of a tumor in subject with an anti-tumor agent or determining the therapeutic utility of a candidate anti-tumor agent, comprising determining the level of Hsp70 in the sample obtained from the subject following administration of the agent to the subject, wherein a reduced level of Hsp70 in the sample of the subject compared to a control indicates progress in the treatment and therapeutic utility of the agent, respectively, or   (i) diagnosing multiple sclerosis (MS) in a subject or whether a MS patient is amenable to the treatment with an anti-multiple sclerosis agent, in particular Hsp70 specific agent, respectively, wherein an elevated level of Hsp70 compared to a control sample is indicative for multiple sclerosis (MS) and possibility for the treatment with the agent, respectively; or   (ii) monitoring the treatment of multiple sclerosis (MS) in subject with an anti-multiple sclerosis agent or determining the therapeutic utility of a candidate anti-neoplastic agent, comprising determining the level of Hsp70 in the sample obtained from the subject following administration of the agent to the subject, wherein a reduced level of Hsp70 in the sample of the subject compared to a control indicates progress in the treatment and therapeutic utility of the agent, respectively.   
     
     
         5 . The method of  claim 1 , wherein the anti-Hsp70 antibody comprises a detectable label, preferably wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, a heavy metal, a tag or a ligand. 
     
     
         6 . The method of  claim 5 , wherein the anti-Hsp70 antibody is biotinylated. 
     
     
         7 . The method of  claim 1 , wherein the level of Hsp70 is determined by ELISA, preferably sandwich ELISA. 
     
     
         8 . The method of  claim 7 , wherein the anti-Hsp70 antibody is used as a detection reagent and antibody against recombinant Hsp70 protein, preferably polyclonal antibody as a capture reagent. 
     
     
         9 . Use of an anti-Hsp70 antibody as defined in  claim 1  for assaying free and exosomal Hsp70 in a test sample, preferably the sample is derived from the body fluid of a subject, preferably wherein the sample comprises serum or plasma. 
     
     
         10 . An assay kit adapted to carry out the method of  claim 1  for determining the presence, progression and/or relapse of a tumor in a subject, comprising means for assaying a serum or plasma sample from the subject for free and liposomal Hsp70 and means for comparing the measured level of Hsp70 to a set of reference standard and/or a control. 
     
     
         11 . An assay kit for determining the presence of free and exosomal Hsp70 in a test sample comprising:
 (i) a detection reagent comprising an anti-Hsp70 antibody as defined in one of  claims 1  to  6 , optionally conjugated to a detectable label, ligand or tag;   (ii) a capture reagent for Hsp70 which is different from the detection reagent of (i);   (iii) optionally a detectable label conjugated to a ligand-binding tag; preferably   (iv) a calibrated immunoassay standard or control of recombinant human Hsp70; and optionally   (v) recommendations for microplates, buffers, diluents, substrates and/or solutions as well as instructions how to perform the assay of any one of  claims 1  to  8 .   
     
     
         12 . The kit of  claim 11 , wherein
 (i) the detection anti-Hsp70 antibody is conjugated to a ligand, preferably biotin;   (ii) the capture reagent comprises a polyclonal antibody directed against human recombinant Hsp70;   (iii) the ligand-binding tag is streptavidin conjugated to an enzyme, preferably horseradish peroxidase (HRP); and/or   (iv) the standard comprises a serial dilution of Hsp 70 including at least a high standard of ≥15 ng/ml, preferably ≥25 ng/ml and more preferably ≥50 ng/ml.   
     
     
         13 . The kit of  claim 10 , comprising:
 (i) a biotinylated detection antibody;   (ii) an antibody-coated ELISA well plate with a capture antibody;   (iii) concentrated HRP-conjugated streptavidin;   (iv) calibrated immunoassay standard recombinant human Hsp70   (v) washing buffer;   (vi) assay/sample diluent buffer(s);   (vii) ELISA colorimetric reagent, preferably 3, 3¢, 5, 5¢-tetramethylbenzidine (TMB) in buffer solution; and   (viii) ELISA stop solution, preferably 0.2 M sulfuric acid.   
     
     
         14 . A therapeutic agent for use in the treatment of a patient suffering from a tumor, characterized in that a serum or plasma sample derived from the patient's blood, compared to a control shows an elevated level of Hsp70 as determined in accordance with the method of  claim 1 , preferably wherein the serum level of Hsp70 is higher than about 7.7 ng/ml, preferably higher than 10 ng/ml. 
     
     
         15 . The method of  claim 1 , the use of  claim 9 , the kit of  claim 10  or the agent of  claim 15 , wherein the tumor is selected from the group consisting of head and neck cancer, lung cancer, colorectal carcinoma, pancreatic cancer, glioblastoma and hematological malignancies or wherein the disorder in which free and lipid-bound (exosomal) heat shock protein 70 (Hsp70) is detected is selected from the group consisting of an infectious or inflammatory disorder.