Method of measuring cholesterol levels by using immunogenic method

Abstract

Disclosed is a method for measuring cholesterol by using a cholesterol dependent cytolysin (CDC), which is a cholesterol binding protein, and an antibody specifically recognizing HDL-C or LDL-C. The method according to the subject matter is capable of rapidly measuring low density lipoproteins and high density lipoproteins at low cost, and thus can be effectively applied in various fields requiring the measurement of low density lipoproteins and high density lipoproteins.

1 . A method of measuring cholesterol level in blood sample in vitro, the method comprising:
 contacting the sample in need of determination of the cholesterol level with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein to form a first complex between a lipoprotein, cholesterol and the CDC;   contacting the first complex with an anti-apolipoprotein antibody to form a second complex comprising the lipoprotein, the cholesterol, the CDC and the antibody; and   detecting the second complex,   wherein the anti-apolipoprotein antibody is two or more distinct antibodies which are distinguishably labelled with different detectable materials.   
     
     
         2 . A method of measuring cholesterol level in blood sample in vitro, the method comprising:
 contacting the sample in need of determination of the cholesterol level with an anti-apolipoprotein antibody to form a first complex between a lipoprotein and the antibody;   contacting the first complex with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein to form a second complex consisting of the lipoprotein, cholesterol, the antibody, and CDC; and   detecting the second complex,   wherein the anti-apolipoprotein antibody is two or more distinct antibodies which are distinguishably labelled with different detectable materials.   
     
     
         3 . The method of  claim 2 , wherein the CDC is selected from the group consisting of ALO (Anthrolysin O) from  Bacillus anthracis ; TLO (Thuringiensilysin O) from  B. thurigiensis ; CLO (Cereolysin O) from  B. cereus ; WLO (Weihenstephanensilysin) from  B. weihenstephanensis ; LLO (Listeriolysin O) from  Listeria monocytogenes ; LSO (Seeligeriolysin O) from  L. seeligeri ; ILO (Ivanolysin) from  L. ivanovii ; SPH (Sphaericolysin) from  Lysinibacillus sphaericus ; ALV (Alveolysin) from  Paenibacillus alvei ; BVL (Brevilysin) from  Brevibacillus brevi ); SLOe (Streptolysin Oe) from  Streptococcus dysgalactiae ; SLO (Streptolysin O) from  S. pyogenes ; SLOc (Streptolysin Oc) from  S. canis ; PSY (Pseudopneumolysin) from  S. pseudonemoniae ; PLY (Pneumolysin) from  S. pneumoniae ; MLY (Mitilysin) from  S. mitis ; SLY (Suilysin) from  S. suis ; ILY (Intermedilysin) from  S. intermedius ; LLY (Lectinolysin) from  S. mitis ; PFO (Perfringolysin O) from  Clostridium perfringens ; BRY (Butyriculysin) from  C. butyricum ; TLY (Tetanolysin O) from  C. tetani ; BLYb (Botulinolysin B) from  C. botulinum  B; BLYe (Botulinolysin E3) from  C. botulinum  E3; BLYc from  C. botulinum  C (Botulinolysin C); NVL (Novyilysin) from  C. novyi ; VLY (Vaginolysin) from Gardenella vaginallis; PLO (Pyolysin) from  Arcanobacterium  pyogenes. 
     
     
         4 . The method of  claim 3 , wherein the CDC is LLO (Listeriolysin O), PFO (Perfringolysin O), PLY (Pneumolysin), or SLO (Streptolysin O) or SLO-4 (Domain 4 of Streptolysin O). 
     
     
         5 . The method of  claim 2 , wherein the anti-apolipoprotein antibody is an anti-LDL antibody or an anti-HDL antibody. 
     
     
         6 . The method of  claim 2 , wherein the blood sample is a whole blood, a plasma or a serum. 
     
     
         7 . The method of  claim 2 , wherein the method is performed by ELISA or a lateral flow assay. 
     
     
         8 . The method of  claim 2 , wherein the antibody is an anti-apolipoprotein B-100 antibody and an anti-apolipoprotein A-1 antibody. 
     
     
         9 . The method of  claim 2 , wherein one of the CDC and the anti-apolipoprotein antibody are fixed on a solid support and the other is labelled with a detectable material. 
     
     
         10 . The method of  claim 9 , wherein the CDC is fixed on a solid support. 
     
     
         11 . The method of  claim 10 , wherein the CDC is fixed on a solid support in advance of contacting the sample with the anti-apolipoprotein antibody. 
     
     
         12 . The method of  claim 9 , wherein the solid support is beads, membranes, slides, or microtiter plates, made of glass, polystyrene, polysaccharide, nylon or nitrocellulose. 
     
     
         13 . The method of  claim 9 , wherein the detectable material is one or more selected from the group consisting of a fluorescent moiety, a chemiluminescent moiety, a bioluminescence moiety, a magnetic particle, an enzyme, a substrate, a radioisotope and a chromophore. 
     
     
         14 . The method of  claim 9 , wherein the detectable material is one or more selected from the group consisting of a radioisotope which is  3 H or  125 I, fluorescent materials, chemiluminescent materials, hapten, biotin, digoxigenin, a horseradish peroxidase, an alkaline phosphatase, and a maleate dehydrogenase. 
     
     
         15 . The method of  claim 2 , wherein the CDC is fixed on a solid support in advance of contacting the sample with the anti-apolipoprotein antibody, and the antibody is an anti-apolipoprotein B-100 antibody and an anti-apolipoprotein A-1 antibody, and the antibodies are labelled with a detectable material. 
     
     
         16 . A kit for measuring cholesterol level in blood comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and an anti-apolipoprotein antibody, wherein the antibody is fixed on a solid support and the CDC is labeled with a detectable material. 
     
     
         17 . The kit of  claim 16  which is a lateral flow kit or ELISA assay kit. 
     
     
         18 . The kit of  claim 16 , wherein the anti-apolipoprotein antibody is an anti-LDL antibody or an anti-HDL antibody. 
     
     
         19 . The kit of  claim 16 , wherein the kit is a lateral flow assay kit, and the CDC is labelled with a detectable material, and the antibody is an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody fixed on a solid support.