Fluorescence polarization method

Abstract

A fluorescence polarization method for analyzing an assay-object in a sample is provided. The fluorescence polarization method includes the steps of: (a) providing a fluorescent-labeled protein in which a protein is covalently bound to a fluorochrome(s), wherein the protein is capable of specifically binding to the assay-object; (b) allowing the fluorescent-labeled protein to bind to the assay-object; and (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescent-labeled protein by its binding to the assay-object.

1 . A fluorescence polarization method for analyzing an assay-object in a sample, the method comprising the steps of: 
 (a) providing a fluorescent-labeled protein in which a protein is covalently bound to a fluorochrome(s), wherein the protein is an antibody, a receptor or an inhibitor, which is capable of specifically binding to the assay-object;    (b) allowing the fluorescent-labeled protein to bind to the assay-object; and    (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescentlabeled protein by its binding to the assay-object.    
     
     
         2 . A fluorescence polarization method according to  claim 1 , wherein the antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a Fab antibody or a (Fab)2 antibody.  
     
     
         3 . A fluorescence polarization method according to  claim 1 , wherein the assay-object is a biological substance, a microorganism, a virus, a pharmaceutical, an environmental pollutant or an abused drug.  
     
     
         4 . A fluorescence polarization method according to  claim 3 , wherein the biological substance is a peptide, a protein, a lipid, a saccharide or a nucleic acid.  
     
     
         5 . A fluorescence polarization method according to  claim 4 , wherein the protein has a molecular weight of 500,000 or more.  
     
     
         6 . A fluorescence polarization method according to  claim 4 , wherein the protein is an antibody, a hormone, an inflammation marker, a coagulation factor, an apolipoprotein, a high density lipoprotein (HDL), a low density lipoprotein (LDL), a glycosylated albumin, a glycosylated hemoglobin, a hemoglobin, a cancer marker or an enzyme.  
     
     
         7 . A fluorescence polarization method according to  claim 6 , wherein the hormone is chorionic gonadotropin, thyroid-stimulating hormone, progesterone, follicular forming hormone, parathyroid-stimulating hormone, adrenocortico-tropic hormone, or insulin.  
     
     
         8 . A fluorescence polarization method according to  claim 6 , wherein the inflammation marker is C-reactive protein (CRP), α1-antitrypsin (α1-AT), α1-antichymotrypsin (α1-X), α1-acid glycoprotein (α1-AG), haptoglobin (Hp), ceruloplasmin (Cp), the 9th component of complement (C9), the 4th component of complement (C4), the 3rd component of complement (C3), complement factor B (B), fibrinogen (Fbg), serum amyloid A (SAA), C1 inhibitor (C1I), a sialoglycoprotein, an acid-soluble protein (ASP) or an immunosuppressive acidic protein (IAP).  
     
     
         9 . A fluorescence polarization method according to  claim 1 , wherein the fluorochrome has a functional group which can bind to a primary, secondary or tertiary amino group, a carboxyl group, a thiol group, a phenyl group, a phenol group or a hydroxyl group.  
     
     
         10 . A fluorescence polarization method according to  claim 1 , wherein a lifetime of fluorescence of the fluorochrome is in the range of 10 nanoseconds to 200 nanoseconds.  
     
     
         11 . A fluorescence polarization method according to  claim 1 , wherein the fluorochrome has a skeletal structure of rhodamine, pyrene, dialkylaminonaphthalene or cyanin.  
     
     
         12 . A reagent for use in a fluorescence polarization method for analyzing an assay-object in a sample, the reagent comprising a fluorescent-labeled protein in which a protein is covalently bound to a fluorochrome(s), wherein the protein is an antibody, a receptor or an inhibitor, which is capable of specifically binding to the assay-object.  
     
     
         13 . A fluorescence polarization method for analyzing a bacteria or a virus in a sample, the method comprising the steps of: 
 (a) providing a fluorescent-labeled antibody in which an antibody is covalently bound to a fluorochrome, wherein the antibody is capable of specifically binding to the bacteria or the virus;    (b) allowing the fluorescent-labeled antibody to bind to the bacteria or the virus; and    (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescent-labeled antibody by its binding to the bacteria or the virus.    
     
     
         14 . A fluorescence polarization method according to  claim 13 , wherein the antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a Fab antibody or a (Fab)2 antibody.  
     
     
         15 . A fluorescence polarization method according to  claim 13 , wherein the bacteria is selected from the group consisting of Rhodospirillaceae, Chromatiaceae, Chlorobiaceae, Myxococcaceae, Archangiaceae, Cystobacteraceae, Polyangiaceae, Cytophagaceae, Beggiatoaceae, Simonsiellaceae, Leucotrichaceae, Achromatiaceae, Pelonemataceae, Spirochaetaceae, Spirillaceae, Pseudomonadaceae, Azotobacteraceae, Rhizobiaceae, Methylomonadaceae, Halobacteriaceae, Enterobacteriaceae, Vibrionaceae, Bacteroidaceae, Neisseriaceae, Veillonellaceae, Organisms oxidizing ammonia or nitrite, Organisms metabolizing sulfer and sulfer compounds, Organisms depositing iron and/or manganese oxides, Siderocapsaceae, Methanobacteriaceae, Aerobic and/or facultatively anaerobic Micrococcaceae, Streptococcaceae, Anaerobic Peptococcaceae, Bacillaceae, Lactobacillaceae, Coryneform group of bacteria, Propionibacteriaceae, Actinomycetaceae, Mycobacteriaceae, Frankiaceae, Actinoplanaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae, Rickettsiaceae, Bartonellaceae, Anaplasmataceae, Chlamydiaceae, Mycoplasmataceae and Acholeplasmataceae.  
     
     
         16 . A fluorescence polarization method according to  claim 13 , wherein the virus is selected from the group consisting of Enterovirus, Cardiovirus, Rhinovirus, Aphthovirus, Calicivirus, Orbivirus, Reovirus, Rotavirus, Abibirnavirus, Piscibirnavirus, Entomobirnavirus, Alphavirus, Rubivirus, Pestivirus, Flavivirus, Influenzavirus, Pneumovirus, Paramyxovirus, Morbillivirus, Vesiculovirus, Lyssavirus, Coronavirus, Bunyavirus, Arenavirus, Human immunodeficiency virus, Hepatitis A virus, Hepatitis B virus and Hepatitis C virus.  
     
     
         17 . A fluorescence polarization method according to  claim 13 , wherein the fluorochrome has a functional group which can bind to a primary, secondary or tertiary amino group, a carboxyl group, a thiol group, a phenyl group, a phenol group or a hydroxyl group.  
     
     
         18 . A fluorescence polarization method according to  claim 13 , wherein a lifetime of fluorescence of the fluorochrome is in the range of 10 nanoseconds to 200 nanoseconds.  
     
     
         19 . A fluorescence polarization method according to  claim 13 , wherein the fluorochrome has a skeletal structure of rhodamine, pyrene, dialkylaminonaphthalene or cyanin.  
     
     
         20 . A reagent according to  claim 12 , wherein the assay-object is a bacteria or a virus, and the specifically-binding protein is an antibody.