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 present application 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.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a national stage application of International Patent Application No. PCT/KR2016/000689, filed Jan. 22, 2016, and claims the benefit of Korean Patent Application Nos. 10-2015-0011355, filed Jan. 23, 2015 and 10-2016-0007850, filed Jan. 22, 2016 in the Korean Intellectual Property Office, the disclosure of which are incorporated herein. 
     
    
     STATEMENT OF SEQUENCE LISTING 
       [0002]    The Sequence Listing submitted in text format (.txt) filed on Jul. 21, 2017, named “SequenceListing.txt”, created on Jul. 17, 2017 (8.51 KB), is incorporated herein by reference. 
       BACKGROUND OF THE INVENTION 
     Field of the Invention 
       [0003]    The present disclosure relates to measuring the cholesterol level, particularly to measuring the cholesterol level based on immunological methods. 
       Description of the Related Art 
       [0004]    Cholesterols are representative blood lipids. It serves as an energy source in the body just like glucose as well as a main source of hormone such as sex and adrenal cortex hormones. For its proper function in the body, they need to move through the circulation in the blood, which requires carriers called lipoprotein. Lipoproteins binding to the cholesterols are divided into LDL(Low Density Lipoprotein) and HDL(High Density Lipoprotein) according to their densities. 
         [0005]    Both LDL and HDL are required for normal function of cells. However when their levels are not in an optimal range (HDL: over 60 mg/dl; and LDL: below 130 mg/dl), it may pose a severe heath problem such as atherosclerosis and thrombus which may lead to a heart attack. Other problems include high blood pressure, stroke, obesity, diabetes, and the like (William P. et al., Incidence of Coronary Heart Disease and Lipoprotein Cholesterol Levels: The Framingham Study., In JAMA, 1986.; M R Law N W, S G Thompson. By how much and how quickly does reduction in serum cholesterol concentration lower risk of ischaemic heart disease, In BMJ, 1994.). 
         [0006]    Thus, the cholesterol levels are in a routine checklist of a blood test in clinical settings, and appropriate measures are taken accordingly to lower the cholesterol level. 
         [0007]    One of the methods to measure the cholesterol level is based on a method called, Friedewald: LDL-c=TC-[HDL-c+TG/k], k=5). However its inaccuracy has been reported in a study involving more than million people (Martin S S et al., J Am Coll Cardiol. 2013 Aug. 20; 62(8):732-9; Larosa J C. J Am Coll Cardiol. 2013 Aug. 20; 62(8):740-1). Friedewald method is particularly inaccurate when the triglyceride level is over 400 mg/dL or lipoproteins called chylomicrons are present abundantly in the blood. Other problems include high cost and time. 
         [0008]    Korean Patent Application Publication 2010-0091176 relates to kits and methods to quantify the cholesterol level of small, dense LDL, and discloses a method and a reagent for scavenging any cholesterol in LDL other than the small, dense LDL in the presence of a phospholipase; and measuring the quantity of a cholesterol in a lipoprotein which remains after the previous step. 
         [0009]    Therefore there exist needs to develop a more convenient and effective method to measure the concentration at a low cost. 
       SUMMARY OF THE INVENTION 
       [0010]    In the present disclosure, there is provided a method to measure the cholesterol level based on CDC (Cholesterol Dependent Cytolysin) and antiboides to cholesterols, which can be advantageously used for rapid and accurate detection of cholesterols. 
         [0011]    It is therefore an aspect of the present disclosure to provide a method to measure the cholesterol level in vitro comprising a step of contacting a sample in need of determination of the cholesterol level with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein by which a first complex between the cholesterol in the sample and the CDC is formed; contacting the first complex with an antibody specifically recognizing the CDC or the cholesterol by which a second complex between the first complex and the antibody is formed; and detecting the second complex. 
         [0012]    Alternatively, the present disclosure provide a method to measure the cholesterol level in vitro comprising contacting a sample in need of determination of the cholesterol level with an antibody specifically recognizing the cholesterol by which a third complex between the cholesterol in the sample and the antibody is formed; contacting the third complex with a CDC (Cholesterol Dependent Cytolysin) specifically recognizing the cholesterol by which a forth complex between the third complex and the CDC is formed; and detecting the forth complex. 
         [0013]    In the foregoing methods, various CDCs having affinity to cholesterols from gram positive bacteria may be employed. 
         [0014]    Still in the foregoing methods, in addition to CDCs, antibodies specifically recognizing the cholesterols are used. Such antibodies include anti-LDSs or anti-HDLs from various types and/or origins specifically recognizing LDL or HDL. 
         [0015]    Still in the foregoing methods, various samples in need of cholesterol measurements include for example, whole blood, plasma or serum. 
         [0016]    Still in the foregoing methods, the present methods may be embodied in various kits such as ELISA or rapid kits based on lateral flow assay. In one embodiment, CDCs are fixed onto a solid support and anti-cholesterol antibodies are labelled with a detectable material to detect a complex, or anti-cholesterol antibodies are fixed onto a solid support and CDCs are labelled with a detectable material to detect a complex. 
         [0017]    In other aspect, there is provided a kit to measure a cholesterol level comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and antibodies to LDL or HDL. 
         [0018]    In another aspect, there is provided a use of CDC (Cholesterol Dependent Cytolysin) and cholesterol antibodies to measure the level of cholesterol. 
       Advantageous Effects 
       [0019]    The present disclosure discloses a method to measure the concentration of the cholesterol using cholesterol binding proteins called CDC (Cholesterol Dependent Cytolysin). The present method enables a rapid and simple determination at a low cost and thus is applicable to various fields of art requiring the quantification of the cholesterol, replacing the previous methods. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  is a schematic diagram showing one embodiment of the present method using CDCs and antibodies to the cholesterol. 
           [0021]      FIG. 2  is a schematic diagram showing another embodiment of the present method using CDCs and antibodies to the cholesterol. 
           [0022]      FIG. 3A  is a result of SDS-PAGE of the antibody to HDL prepared in one embodiment of the present disclosure. 
           [0023]      FIG. 3B  is a result of measuring the concentration of HDL antibody of  FIG. 3A . 
           [0024]      FIG. 4A  is a result of SDS-PAGE of the antibody to LDL prepared in one embodiment of the present disclosure. 
           [0025]      FIG. 4B  is a result of measuring the concentration of LDL antibody of  FIG. 4A . 
           [0026]      FIG. 5  shows a nucleic acid sequence of a PLY (SEQ ID NO:1) prepared in the present disclosure as one example of CDC and a result of SDS-PAGE of a purified recombinant PLY expressed and isolated in one embodiment of the present disclosure. 
           [0027]      FIG. 6  shows a nucleic acid sequence of a PFO (SEQ ID NO:2) prepared in the present disclosure as one example of CDC and a result of SDS-PAGE of a purified recombinant PFO expressed and isolated in one embodiment of the present disclosure. 
           [0028]      FIG. 7  shows a nucleic acid sequence of a LLO (SEQ ID NO:3) prepared in the present disclosure as one example of CDC and a result of SDS-PAGE of a purified recombinant LLO expressed and isolated in one embodiment of the present disclosure. 
           [0029]      FIG. 8  shows a nucleic acid sequence of a SLO_D4 (SEQ ID NO:4) prepared in the present disclosure as one example of CDC and a result of SDS-PAGE of a purified recombinant SLO_D4 expressed and isolated in one embodiment of the present disclosure. 
           [0030]      FIG. 9  shows a nucleic acid sequence of a SLO (SEQ ID NO:5) prepared in the present disclosure as one example of CDC and a result of SDS-PAGE of a purified recombinant SLO expressed and isolated in one embodiment of the present disclosure. 
           [0031]      FIG. 10  is a result of measuring the concentration of HDL-C in which cholesterols were bound with CDC (SLO, SLO_D4, LLO, PFO, or PLY) and detected using the antibody #11C1 which recognizes Apo A-I, main components of HDL-C. 
           [0032]      FIG. 11  is a result of measuring the concentration of LDL-C in which cholesterols were bound with CDC (SLO, SLO_D4, LLO, PFO, or PLY) and detected using the antibody #4C2 which recognizes Apo B-100, a main components of LDL-C. 
           [0033]      FIG. 12  is a graph showing the correlation of the data generated from two different types of readers, in which the cholesterol concentrations were measured from blood by the rapid kit using CDC protein and LDL antibody prepared in the present disclosure. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0034]    The present disclosure described here is based, in part, on the discovery that the concentration of cholesterol can be determined in a sample by use of CDC (Cholesterol Dependent Cytolysin) and antibodies to LDL or HDL. 
         [0035]    CDCs (Cholesterol Dependent Cytolysin) employed herein belong to a family of toxic proteins that have a beta-barrel form and are secreted from gram positive bacteria. They function in cells by binding to a target cell and inserting themselves having a beta-barrel form into the cell membrane to make a hole through the membrane. It is the cholesterol present on the cell membrane that is required for pore formation. CDCs generally show a strong affinity to cholesterols on the cell membrane. 
         [0036]    In the present discourse, methods to determine the concentration of cholesterol in a sample have been developed by unexpected efforts. 
         [0037]    It is therefore an aspect of the present invention to provide a method of measuring the cholesterol level in vitro comprising: contacting a sample in need of determination of the cholesterol level with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein by which a first complex between the cholesterol in the sample and the CDC is formed; contacting the first complex with an antibody specifically recognizing the CDC or the cholesterol by which a second complex between the first complex and the antibody is formed; and detecting the second complex. 
         [0038]    In accordance of the present methods, a sample in need of determination of the cholesterol level is reacted with CDC to form a first complex as a first step. Then the first complex is reacted with an antibody specifically recognizing the cholesterol. Then the concentration of the cholesterol is determined by detecting the second complex using an antibody specifically recognizing either the CDC or the cholesterol. 
         [0039]    The CDCs which may be employed in the present disclosure include various proteins from gram positive bacteria having affinity to cholesterol. Examples of such may include, but are not limited to, the ones as disclosed below, the sequence of which is known and can be accessed from a public database for example with a number provided in a parenthesis: ALO (Anthrolysin O) from  Bacillus anthracis  (for example UniProt Accession NO: Q81N62); TLO (Thuringiensilysin O) from  B. thurigiensis  (for example NCBI Accession NO: YP_037419); CLO (Cereolysin O) from  B. cereus  (for example NCBI Accession NO: YP_002369889.1); WLO (Weihenstephanensilysin) from  B. weihenstephanensis  (for example NCBI Accession NO: ABY46062), LLO (Listeriolysin O) from  Listeria monocytogenes  (for example NCBI GenBank Accession NO: ABH07645); LSO (Seeligeriolysin O) from  L. seeligeri  (for example Uniprot Accession NO: P31830.1); ILO (Ivanolysin) from  L. ivanovii  (for example NCBI GenBank Accession NO: AAR97343.1); SPH (Sphaericolysin) from  Lysinibacillus sphaericus  (for example NCBI Accession NO: YP_001699692.1); ALV (Alveolysin) from  Paenibacillus alvei  (for example Uniprot Accession NO: P23564); BVL (Brevilysin) from  Brevibacillus brevis  (for example NCBI Accession NO: YP_002770211.1); SLOe (Streptolysin Oe) from  Streptococcus dysgalactiae  (for example NCBI GenBank Accession NO: BAD77791); SLO (Streptolysin O) from  S. pyogenes  (for example NCBI Accession NO: NP_268546.1); SLOc (Streptolysin Oc) from  S. canis  (for example Uniprot Accession NO: Q53957); PSY (Pseudopneumolysin) from  S. pseudonemoniae  (for example NCBI GenBank Accession NO: ACJ76900); PLY (Pneumolysin) from  S. pneumoniae  (for example NCBI GenBank Accession NO: ABO21366.1); MLY (Mitilysin) from  S. mitis  (for example NCBI GenBank Accession NO: ABK58695); SLY (Suilysin) from  S. suis  (for example NCBI GenBank Accession NO: ABE66337.1); ILY (Intermedilysin) from  S. intermedius  (for example NCBI GenBank Accession NO: BAE16324.1); LLY (Lectinolysin) from  S. mitis  (for example NCBI GenBank Accession NO: BAE72438.1); PFO (Perfringolysin O) from  Clostridium perfringens  (for example NCBI Accession NO: NP_561079); BRY (Butyriculysin) from  C. butyricum  (for example NCBI Accession NO: ZP_02950902.1); TLY (Tetanolysin O) from  C. tetani  (for example NCBI Accession NO: NP_782466.1); BLYb (Botulinolysin B) from  C. botulinum B  (for example NCBI Accession NO: YP_001886995.1); BLYe (Botulinolysin E3) from  C. botulinum  E3 (for example NCBI Accession NO: YP_001921918.1); BLYc from  C. botulinum C  (Botulinolysin C) (for example NCBI Accession NO: ZP_02620972.1); NVL (Novyilysin) from  C. novyi  (for example NCBI Accession NO: YP_878174.1); VLY (Vaginolysin) from  Gardenella vaginallis  (for example UniProt Accesion NO: B2YGA4), PLO (Pyolysin) from  Arcanobacterium pyogenes  (for example NCBI GenBank Accession NO: AAC45754.1). 
         [0040]    CDCs have a characteristic of adhering to the cell membrane and of entering inside of the cells causing a cell lysis by making a pore through the membrane. It is the characteristic of CDCs&#39; binding affinity to cholesterol that is utilized in the present methods. Thus various CDCs exemplified as above in addition to LLO, PFO, PLY, and SLO used herein can also be employed in the present methods. 
         [0041]    CDC proteins can be prepared by genetic recombination methods known in the art including for example amplification of a gene of interest followed by expression in bacteria or by methods described in the Examples of the present disclosure. 
         [0042]    The present methods can be used for determining the cholesterol level from various samples that require the measurement, which includes for example whole blood, plasma, or serum from mammals, particularly human beings without being limited thereto. 
         [0043]    In other exemplary embodiment of the present disclosure, samples which may be employed in the present methods include any samples containing or expected to contain cholesterol, for example whole blood, plasma, or serum without being limited thereto. 
         [0044]    The cholesterols that are measured by the present methods are insoluble in aqueous environment and thus require a carrier protein called lipoprotein to move through the circulation in the blood. The lipoproteins which bind to cholesterol for its movement are divided into LDL(Low Density Lipoprotein) and HDL(High Density Lipoprotein). Both LDL and HDL concentrations are needed to be measured in clinical settings. 
         [0045]    In the present methods, each concentration of LDL and HDL can be measured by reacting the CDC complexed with cholesterol with antibody specific to LDL or HDL, respectively. 
         [0046]    Alternatively, in the present methods, the concentration can be measured by reacting the complex of CDC and cholesterol with antibody specific to CDC. 
         [0047]    In one embodiment of the present disclosure, CDCs form a first complex by binding to cholesterols, and then LDL or HDL in the first complex are reacted with each specific antibody to LDL or HDL to form a second complex. Then the second complex is detected for determination of the concentration of LDL and HDL. 
         [0048]    Alternatively, in other embodiment of the present disclosure, the cholesterols in a sample are firstly combined with antibodies specific to each of LDL and HDL to form a third complex. Then the third complex is bound to CDC to form a forth complex, which is then detected to determine the concentration of LDL and HDL. 
         [0049]    The detection of a second or a forth complex may be achieved using various methods known in the art. For example, the concentration may be determined directly by reading the signal generated from a labelling material conjugated to the antibody specific to cholesterols or indirectly by reading the signal from a separate antibody labelled with chromophores, the separate antibody recognizing the antibody specific to cholesterols. 
         [0050]    In this perspective, there is provided a method to detect or measure the concentration of cholesterol in vitro, which comprises a step of contacting a sample in need of cholesterol measurement with an antibody specifically recognizing the cholesterol whereby a third complex between the cholesterol and antibody is formed; a step of contacting the third complex with CDC (Cholesterol Dependent Cytolysin) specifically binding to the cholesterol whereby a forth complex between the third complex and CDC is formed; and a step of detecting the forth complex to determine the concentration. 
         [0051]    The present methods disclosed herein may be embodied by use of various methods based on immunological reactions. 
         [0052]    The antibodies which may be employed in the present disclosure specifically recognize LDL, HDL, or Apo proteins comprised in the LDL and HDL. For example, in case of LDL, antibody specific for Apo 100 may be used. These antibodies are commercially available or may be prepared by methods known in the art. For example, the antibodies of interest may be prepared by the methods disclosed in the present Examples. Also the antibodies which may be used in the present disclosure include monoclonal or polyclonal antibodies. Further the antibodies which may be used in the present disclosure include full length or partial length of antibodies, aptamers, avidity multimers or peptidomimetics. 
         [0053]    In one embodiment of the present disclosure, ELISA (enzyme-linked immunosorbent assay) is used. ELISA is a method to quantify or detect a particular components (antigen) present in minute amount (generally below nanogram) in biological samples. For example, proteins with specific affinity to the component of interest are conjugated to inert polymers and biological samples are applied thereto to form a complex between the component of interest in the sample and the proteins, after which the complex is detected by using a proper antibody. The detection antibodies are conjugated with an enzyme as described hereinafter able to catalyze a reaction in the presence of appropriate substrate to generate a signal such as light or a color change. The signals generated are then detected and the concentrations of the components of interest are determined accordingly. In this case, CDCs are fixed to a polymer (or a solid support) and cholesterols are applied thereto to form a complex between the CDC and cholesterols. The complex is then detected by antibodies to LDL or HDL which may be labelled. Or additional labelled antibodies recognizing the antibodies complexed with LDL or HDL may be used. In other example, LDL or HDL antibodies may be fixed to an inert polymer and the biological sample is applied thereto. Then antibodies specific to CDCs are applied thereto after the unbound sample is removed. 
         [0054]    In accordance with the present disclosure, CDC, or HDL or LDL antibodies may be attached to a solid support or labelled with a proper material to aid detection, depending on particular methods employed. The solid supports have an inert surface on which CDCs or antibodies can be attached, and include but are not limited to, beads, membranes, slides or microtiter plates made of glass, plastic (for example polystyrene), polysaccharide, nylon or nitrocellulose. 
         [0055]    CDCs or antibodies in accordance with the present disclosure may be labelled with proper labeling material, which can be detected. The labeling materials refer to materials that can generate signals detectable by proper methods such as spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and/or immunological methods. Such materials include for example a fluorescent moiety, a chemiluminescent moiety, a bioluminescence moiety, a magnetic particle, an enzyme, a substrate, a radioisotope and a chromophore. 
         [0056]    CDCs or antibodies in accordance with the present disclosure may be labelled with labeling materials enabling direct or indirect detection such as, for example radioisotopes such as  3 H or  125 I, fluorescent materials, chemiluminescent materials, hapten, biotin, digoxigenin and the like for quantitative or qualitative detection. Or in the present methods, the quantitative or qualitative detection may be performed by using antibodies conjugated with a enzyme such as a horseradish peroxidase, an alkaline phosphatase, or a maleate dehydrogenase, which generates a color change or light upon reaction in the presence of a proper substrate. The quantitative or qualitative determination of the component of interest in the sample can then be determined from the amount of antibodies or CDCs, which is measured by the intensity of the signal from the light or color. For example, Biochemistry, 2nd edition, B. D. Hames and N. M. Hooper, Springer-Verlag New York 2000, pages 112-114 may be referred for further details. 
         [0057]    In other embodiment of the present disclosure, a rapid kit based on lateral flow assay is used. In this case, CDC, or LDL or HDL antibodies are attached to the surface of a solid substrate, which for example is provided as glass slides or nitrocellulose membranes. Rapid kits based on lateral flow assay are known in the field of POCT (Point of care test) art, in which CDCs are attached to a solid support such as nitrocellulose membrane and then the reaction mixture of a sample such as serum and HDL or LDL antibodies is contacted with the membrane by applying the mixture at one end of the membrane. By such contact, the mixture moves through the membrane by a capillary action during which cholesterols in the sample bind to CDCs fixed to the surface of the solid substrate and the concentration of cholesterols are determined by detecting the signals generated from the labeling materials conjugated to the antibodies bound to the cholesterols. 
         [0058]    In other aspect, the present disclosure provides kits for cholesterol measurement comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and LDL antibody and/or HDL antibody. The components comprised in the present kits and the methods which may be used with the present kit are as described hereinbefore and the Examples. 
         [0059]    The present disclosure is further explained in more detail with reference to the following examples. These examples, however, should not be interpreted as limiting the scope of the present invention in any manner. 
       EXAMPLES 
     Materials 
       [0060]    Antibody production and purification: female BALB/c mice of 6-8 weeks were used. Human HDL and LDL were purchased from ProSpec (USA), complete Freund&#39;s adjuvant and Freund&#39;s incomplete adjuvant were purchased from MP Biomedicals Inc (USA). Fetal bovine serum (FBS) and Dulbecco&#39;s Modified Eagle Medium(4.5 g/L DGlucose, L-Glutamine, 110 mg/L sodium pyruvate), HAT supplement, and HT supplement, Antibiotics were purchased from Gibco (USA). PEG1500 (Polyethylene Glycol 1500) were purchased from Roche (Swiss). The medium contained 10% FBS. Pristane (2,6,10,14-tetrametheylepentadecane) and Protein G Immobilized on agarose were purchased from Sigma (USA). 
         [0061]    For isotyping, Pierce™ Rapid Mouse Antibody Isotyping Kits (Quickly determine mouse monoclonal antibody (MAb) class and subclass identity.) were used. 
         [0062]    For ELISA, anti-mouse IgG-HRP from KPL (USA) was used as a secondary antibody. TMB/E solution was purchased from EMD Millipore Corporation (USA). Streptavidin-HRP was purchased from R&amp;D systems (USA). The blocking buffer were prepared by adding 1% BDA in 0.1% PBS/T. 
         [0063]    For cartridge preparation and fluorescent conjugation, NC membrane HFB13504 from Millipore was used. As a sample pad, Whatman 903 was used. As an absorbent pad, TN415 from Woorichem (Korea). FPR-648 from BioActs (Korea) was used for fluorescent conjugation. DDB contains 1% BSA, 0.1% Tween-20, 0.1% NaN3 in 0.1% PBST pH 7.4. DB was prepared by adding 1 μg/ml anti-LDL #4c2, and 50 ng/ml anti-DNP #3A6G9 to DDB. 
       EXAMPLE 1. Preparation of HDL and LDL Antibodies 
     1-1. Immunization 
       [0064]    To induce an immunization reaction in mice, the LDLs were intraperitoneally (I.P.) injected into the mice. For a first immunization, 200 μg/200 μl of human LDL and HDL were mixed with 100 μl of complete Freund&#39;s adjuvant, and the mixture were intraperitoneally injected into BALB/c mice at 300 μl/mouse. After 10 days of the first injection, the mixture of HDL, LDL and Freund&#39;s incomplete adjuvant was injected every 10 days for a total of 3-4 times. For a cell fusion, 500 μg/300 μl of HDL and LDL without the adjuvant was intraperitoneally injected. 
       1-2. Feeder Cell Preparation 
       [0065]    One day before fusing cells, BALB/c mice of 7-8 weeks of age were sacrificed under CO 2  and the coat was removed. Then 5 ml of 11.6% sucrose solution was intraperitoneally injected and the site of injection was massaged for 1 min. Then the sucrose solution with macrophage were recovered from the peritoneal cavity using syringe, which were transferred to a tube and centrifuged at 1500 rpm for 1 min to collect feeder cells. The feeder cells were then suspended in HAT complete DMEM and seeded onto each well of five 96 well plates at 140 μl/well and incubated overnight at 37° C. CO 2  incubator. 
       1-3. Cell Fusion 
       [0066]    This example describes a process to prepare B cells, an immune cell. The spleen was removed from the mice immunized with HDL and LDL. The spleen was then placed onto a culture dish containing incomplete DMEM and dissected into smaller pieces, which was then transferred to a tube and precipitated for 5 min to collect the supernatant. 
         [0067]    Then SP2/O cells were used as myeloid cells, which were cultured in 75T cm 2  flask until at least 75% confluency. The cells were then centrifuged at 1500 rpm for 5 min for precipitation. The precipitated cells were then mixed with B cells and centrifuged at 1500 rpm for 1 min. The precipitated mixture of cells were then dispersed and 1 ml of PEG1500 (Polyethylene Glycol 1500) was added thereto in small portions for 90 seconds while shaking the tube for improving the fusion. Then the tube was shaken again for an additional 90 seconds and complete DMEM (40 ml) was added slowly for 10 min, which was then centrifuged for 1 min at 1500 rpm. The precipitated cells were then suspended in HAT complete DMEM and seeded onto a plate with feeder cells at 140 μl/well and incubated in a 37° C. CO 2  incubator. 
       1-4. Screening 
       [0068]    For a first screening, when the colonies were formed after the cell fusion, the medium was removed and 280 μl/well of fresh HAT complete DMEM was added. When the colonies were grown big enough, indirect ELISA was used for screening. 1μg/ml of HDL, LDL was added at 50 μl/well and incubated for 2 hours at 37° C. Then each well was washed with 1XPBST 3 times and then blocking buffer was added to each well at 200 μl/well followed by an incubation at 37° C. for 1 hour. Then the wells were washed with 0.1% PBST 3 times and 50 μl/well of medium with fused cells was added as a first antibody and incubated at 37° C. for 1 hour. Then the wells were washed with 0.1% PBST for 3 times and as a second antibody 200 ng/ml of anti-goat mouse IgG HRP was added at 50 μl/well and incubated at 37° C. for 1 hour. Then the wells were washed with 0.1% PBST 3 times and 100 μl/well of TMB/E solution was added to each well and incubated for 15 min at RT. Then the absorbance of each well was measured at 630 nm using ELISA reader and only the wells having absorbance of 1.0 and above were selected for further screening. The cells from the selected wells were then transferred to each well of 24 well plate and cultured in HAT complete DMEM in 37° C. CO 2  incubator. The cells were monitored and a second screening was performed as described in the first screening when the cells were fully grown. The cells of a well having O.D. value of 1 or above at 650 nm were transferred to each well of 6 well plate. By this way, 3 rd  and 4 th  screenings were performed and the screened cells were transferred to 75T cm 2  flask. The cells were sub-cultured and the parent cells were stored frozen. 
       1-5. Cloning and Subculture 
       [0069]    Homogenous clones of the cells producing antibody were isolated through cloning process as described below. As a first step, the feeder cells were seeded onto each well of 96 well plate and incubated overnight in a 37° C., CO 2  incubator. Next the parent cells were counted and diluted to include one cell in 140 μl, which was added to each well and incubated in a 37° C., CO 2  incubator to obtain a colony. Then indirect ELISA was used to select monoclonal cells with higher value and successively subcultured from 96 well, to 24 well, to 6 well plate, to 25 cm 2  T flask and finally to 75 cm 2  T flask. 
       1-6. Mouse Experiment 
       [0070]    BALB/C was injected with pristine at 300 μl/mouse to disable the immune system. After 7 days, the fused cells were diluted in incomplete DMEM at the concentration of  1 × 10   ̂ 6, which were then intraperitoneally injected into mice at 300 μl/mouse. The ascites fluid generated after the injection was harvested with a syringe from the mice anesthetized with CO 2  inhalation. The ascite harvested then was centrifuged at 3500 rpm for 15 min to remove tissues and lipids and the like and to collect the supernatant, which was stored frozen at −20° C. until use. 
       1-7. Purification of Monoclonal Antibodies and Confirmation 
       [0071]    The frozen ascite prepared above was melted at RT and centrifuged at 12000 rpm for 40 min to remove any residual lipids. Then 55% ammonium sulfate was added to the supernatant and dissolved, which were incubated overnight at 4° C. and centrifuged at 15000 rpm for 40 min to collect precipitates. The precipitates were dissolved in the same volume of 1× PBS (pH 7.4) as that of the ascite and desalted by dialysis. For dialysis, 1× PBS pH 7.4 was used as a buffer and the buffer was changed every 3 hours for a total of 4 times. The dialyzed ascite was diluted 5 times with 1× PBS pH 7.4 and applied to a column filled with protein-G to which the proteins are binding. The unbound proteins were removed by washing the column with 1× PBS pH 7.4. Then 100 mM glycine buffer pH 2.5 was applied to the column for elution in 3 ml aliquots. 1M Tris-Cl pH 8.0 was used as a neutralization buffer. 
         [0072]    To remove glycine, a dialysis in 1× PBS pH 7.4 was performed. The purified antibody was confirmed to consist of a heavy chain 50-55kDa and a light chain 20-25kDa in size in a SDS-PAGE gel, which was then used for ELISA. To each well of 96 well plate, HDL, LDL of 1 μg/ml was added at 50 μl/well and the plate was incubated at 37° C. for 2 hours followed by washing 3 times with 0.1% PBST and blocked for 1 hour in 200 μl/well of blocking buffer at 37° C. Then the plate was washed 3 times with 0.1% PBST and incubated with 50 μl/well of the 1 st  antibody prepared by two-fold serial dilution starting from 1 μl/ml at 37° C. for 1 hour. Then the plate was washed 3 times with 0.1% PBST and incubated with 50 μl/well of anti-goat mouse IgG HRP of 200 ng/ml as a second antibody at 37° C. for 1 hour. Then the plate was washed 3 times with 1× PBST and incubated with 100 μl/well of TMB/E for 15 min at RT followed by reading at O.D. 650 nm using an ELISA Reader. Results are shown in  FIGS. 3 and 4 , which confirm that the purified antibodies of the present disclosure have both a light and a heavy chain and specifically recognize each corresponding antigen. 
       EXAMPLE 2. Preparation of CDC (Cholesterol Dependent Cytolysin) 
     2-1. Preparation of Pneumolysin (PLY) 
       [0073]    Pneumolysin (PLY) is a protein produced from  Streptococcus pneumoniae  and has an amino acid sequence appropriate for production of recombinant PLY (NCBI ID: WP_001284361.1), which was used for codon optimization to generate an optimized nucleotide sequence encoding the same. The polynucleotide having an optimized codon sequence was then synthesized from Bioneer (Korea). 
         [0074]    The gene was then amplified by PCR and cloned into BamHI/XhoI site of pET21a (Novagen), which was then transformed into a host cell BL-21™ DE3 Star to select a clone expressing rPLY. pET-21a.ply_BL21™ DE3 star cells were grown in O/N and subcultured in 4.2 L LB medium containing Amp+ cam at 37° C. with shaking at 200 rpm. When 0.D value has reached 0.5, the culture was induced at 30° C. for 4 hours in the presence of IPTG at the final concentration of 20 mM, Then the cells were harvested. The cells were then resuspended in 150 ml of native purification buffer containing protease, DNase and RNase. Then cells were lysed by sonication 10 times under 10 sec sonication and 10 sec rest cycle at 80% amplitude. The cell lysates were centrifuged for 30 min at 10000 rpm. 10 ml of 50% TALON resin (Clonetech) was applied to a purification column of 20 ml in size. The resins were completely stabilized (5-10 min) and eluted. 40 ml of sterilized water was used to resuspend the resin by tapping the colum in upside down position. Then 10 ml of Native purification Buffer (50 mM NaH2PO4, pH 8.0; 0.5 M NaCl) was used to resuspend the resin by tapping the colum in upside down position. After stabilizing the resin, the supernatants were decanted. The process was repeated one more time. 
         [0075]    150 ml of lysates were prepared in a native condition and replaced with 10 mM imidazole. The lysate was then mixed with the prepared resin on a roller mixer for 1 hour. The resin was then transferred to the column and stabilized. The flow obtained was again transferred to the stabilized column and the fractions were collected. 
         [0076]    30 ml Native Wash Buffer (50 Mm NaH2PO4, pH 8.0; 0.5 M NaCl, 20 mM imidazole) was applied to the column and eluted 5 times. For a SDS-PAGE analysis, the supernatant was stored at 4° C. The column was eluted with 20 ml of Native Elution Buffer (50 mM NaH2PO4, pH 8.0; 0.5 M NaCl, 250 mM imidazole) and subjected to a filtration using centricon ultra-filtration (10,000 MWCO) exchanged with PBS(G+E, pH 7.4). As shown in  FIG. 5 , recombinant Pneumolysin was successfully expressed, and purified under a native condition. 
       2-2. Preparation of Perfringolysin O (PFO) 
       [0077]    Perfringolysin O (PFO) is a protein produced from  Clostridium perfringens  and has an amino acid sequence appropriate for production of recombinant PFO (NCBI ID: WP_003467630.1), which was used for codon optimization to generate an optimized nucleotide sequence encoding the same. The polynucleotide having an codon optimized sequence was then synthesized from Bioneer (Korea). 
         [0078]    The gene was then amplified by PCR and cloned into a BamHI/XhoI of pET21a (Novagen), which was then transformed into a host cell BL-21™ DE3 Star to select a clone expressing rPFO. pET-21a.pfo_BL-21 DE3 star cells were grown in O/N and subcultured in 5.4 L LB medium containing Kan+ cam at 37° C. with shaking at 200 rpm. When the 0.D value has reached 0.5, the culture was induced at 30° C. for 4 hours in the presence of IPTG at the final concentration of 0.2 mM. Then the cells were harvested. The cells were then resuspended in 150 ml of native purification buffer containing protease, DNase and RNase. Then cells were lysed by sonication 10 times under 10 sec sonication and 10 sec rest cycle at 80% amplitude. The cell lysates were centrifuged for 30 min at 10000 rpm. 10 ml of 50% TALON resin (Clonetech) was applied to a purification column of 20 ml in size. The resins were completely stabilized (5-10 min) and eluted. 40 ml of sterilized water was used to resuspend the resin by tapping the colum in upside down position. The resin was stabilized before the supernatant was decanted. Then 10 ml of native purification Buffer (50 mM NaH2PO4, pH 8.0; 0.5 M NaCl) was used to resuspend the resin by tapping the colum in upside down position. After stabilizing the resin, the supernatants were decanted. The process was repeated one more time. 
         [0079]    150 ml of the lysates were prepared in a native condition and replaced with 10 mM imidazole. The lysate was then mixed with the prepared resin on a roller mixer for 1 hour. The resin was then transferred to the column and stabilized. The flow obtained was again transferred to the stabilized column and the fractions were collected. 30 ml Native Wash Buffer (50 Mm NaH2PO4, pH 8.0; 0.5 M NaCl, 20 mM imidazole) was applied to the column and eluted 5 times. For a SDS-PAGE analysis, the supernatant was stored at 4° C. The column was eluted with 20 m1 of Native Elution Buffer (50 mM NaH2PO4, pH 8.0; 0.5 M NaCl, 250 mM imidazole) and subjected to a filtration using centricon ultra-filtration (10,000 MWCO) exchanged with PBS(G+E, pH 7.4). As shown in  FIG. 6 , recombinant Perfringolysin 0 was successfully expressed, and purified under a native condition. 
       2-3. Purification of Listeriolysin O (LLO) 
       [0080]    Listeriolysin O (LLO) is a protein produced from  Listeria monocytogenes  and has an amino acid sequence appropriate for production of recombinant LLO (NCBI ID: WP_003722731.1), which was used for codon optimization to generate an optimized nucleotide sequence encoding the same. The polynucleotide having an codon optimized sequence was then synthesized from Bioneer (Korea). 
         [0081]    The gene was then amplified by PCR and cloned into a BamHI/XhoI of pET21a (Novagen) as shown in upper portion of  FIG. 7 , which was then transformed into a host cell Codon plus (RIPL) to select a clone expressing rLLO. pET-21a.LLO_Codon Plus (RIPL) cells were grown in O/N and subcultured in 5.4 L LB medium containing Amp+ cam at 37° C. with shaking at 200 rpm. When the O.D of the cells has reached 0.5, the culture was induced at 18° C. with 0.2 mM IPTG. Then the cells were harvested. The cells were then resuspended in 150 ml of native purification buffer containing protease, DNase and RNase. Then cells were lysed by sonication 10 times under 10 sec sonication and 10 sec rest cycle at 80% amplitude. The cell lysates were centrifuged for 30 min at 12000 rpm. 5 ml of 50% TALON resin (Clonetech) was applied to a purification column of 20 ml in size. The resins were completely stabilized (5-10 min) and eluted. 10 ml of sterilized water was used to resuspend the resin by tapping the column in upside down position. The resin was stabilized before the supernatant was decanted. Then 5 ml of native purification Buffer (50 mM NaH 2 PO 4 , pH 8.0; 0.5 M NaCl) was used to resuspend the resin by tapping the column in upside down position. After stabilizing the resin, the supernatant was decanted. The process was repeated one more time. 
         [0082]    20 ml of the lysates were mixed with the resin prepared in a native condition on a roller mixer for 1 hour. The resin was then transferred to the column and stabilized. The flow obtained was again transferred to the stabilized resin and the fractions were collected. 15 ml Native Wash Buffer (50 Mm NaH 2 PO 4 , pH 8.0; 0.5 M NaCl, 20 mM imidazole) was applied to the column and eluted 5 times. For a SDS-PAGE analysis, the supernatant was stored at 4° C. The column was eluted with 10 ml of Native Elution Buffer (50 mM NaH 2 PO 4 , pH 8.0; 0.5 M NaCl, 250 mM imidazole) and subjected to a filtration using centricon ultra-filtration (10,000 MWCO) exchanged with PBS(G+E, pH 7.4). As shown in  FIG. 7 , recombinant Listeriolysin O (LLO) was successfully expressed, and purified under a native condition. The yield was appropriate for the protocol described above to be used in a production of LLO in large scale. 
       2-4. Expression and Purification of SLO D4 
       [0083]    SLO consists of 4 domains and the domain 4 at N-terminus contains SH− group that binds to cholesterol. Thus, domain 4 of SLO was selected and prepared as a recombinant protein in consideration of the 3D structure of SLO, in which SH- group may not be exposed. 
         [0084]    The nucleotide sequence encoding the domain 4 was codon optimized and synthesized by GeneArt® (Invitrogen) and cloned into pET100. The cloned vector was then transformed into a host cell  E. coli,  BL-21 Star to select a clone expressing rSLO D4. 
         [0085]    pET100.SLO D4-BL-21 star cells were grown in O/N and subcultured in 4.2 L LB medium containing Amp+ cam at 37° C. with shaking at 200 rpm. When the O.D of the cells has reached 0.5, the culture was induced at 30° C. for 4 hours in the presence of 0.2 mM IPTG. Then the cells were harvested. The cells were then resuspended in 150 ml of native purification buffer containing protease, DNase and RNase and were harvested. Then cells were lysed by sonication 10 times under 10 sec sonication and 10 sec rest cycle at 80% amplitude. The cell lysates were centrifuged for 30 min at 10000 rpm. 10m1 of 50% TALON resin (Clonetech) was applied to a purification column of 20 ml in size by pipetting. The resins were completely stabilized (5-10 min) and eluted. 100 ml of sterilized deionized water was used to resuspend the resin by tapping the colum in upside down position. The resin was stabilized before the supernatant was decanted. Then 10 ml of native purification buffer (50 mM NaH 2 PO 4 , pH 8.0; 0.5 M NaCl) was used to resuspend the resin by tapping the colum in upside down position. After stabilizing the resin, the supernatant was decanted. The process was repeated one more time. 
         [0086]    150 ml of the lysates were prepared in a native condition and replaced with imidazole. Then the lysates were mixed with the prepared resin on a roller mixer for 1 hour. The resin was then transferred to the column and stabilized. The flow obtained was again transferred to the stabilized resin and the fractions were collected. 40 ml Native Wash Buffer (50 Mm NaH 2 PO 4 , pH 8.0; 0.5 M NaCl, 20 mM imidazole) was applied to the column and eluted 5 times. For a SDS-PAGE analysis, the supernatant was stored at 4° C. The column was eluted with 20 ml of Native Elution Buffer (50 mM NaH 2 PO 4 , pH 8.0; 0.5 M NaCl, 250 mM imidazole) and subjected to a filtration using centricon ultra-filtration (5,000 MWCO) exchanged with PBS(G+E, pH 7.4). As shown in  FIG. 8 , recombinant SLO D4 was successfully expressed, and purified under a native condition. 
       2-5. Expression and Purification of SLO 
       [0087]    As shown in  FIG. 9 , a gene encoding SLO recombinant protein was amplified by PCR and the PCR product was cloned into pET21a vector, which was used to transform  E. coli . The cells were then grown in LB medium at 37° C. with shaking at 250 rpm. When the O.D. of the cells has reached 0.5-0.7, IPTG was added to the culture at the final concentration of 1 mM and induced for 3 hours. Then cells were harvested by centrifuge and the pellets were lysed in lysis buffer(10 mM sodium phosphate, 0.5M NaCl, 10 mM 2-mercaptoethanol, 10 mM EDTA, pH 7.0 with NaOH) in the presen of protease inhibitor using a supersonic device (SONICS and MATERIALS INC, USA). Then the expression was confirmed by SDS-PAGE gel analysis. The proteins were purified by exchange chromatography method. Results are shown in  FIG. 9 . 
         2 - 6 . Labelling of SLO D4 Protein 
       [0088]    Protein and biotin were mixed at the concentration ratio of 10:1 (100 μl) and 1M sodium bicarbonate was added to the mixture at 1/10 of the total voume (10 μl), which was then incubated overnight at 4° C. To pack lm of DEAE sephadex™ G-25, resins were placed in a column and centrifuged at 3000 rpm for 20 min to remove buffer. Then biotinylated SLO was put into the column and centrifuged at 2000 rpm for 20 min. 
         [0089]    ELISA was performed to confirm the biotinylation of rSLO. SLO-biotin was prepared by two-fold serial dilution starting from 2 μg/ml and added to each well of 8 well strip at 5 μl/well. The wells were then incubated at 37° C. for 2 hours and washed 3 times with 1×PBST followed by blocking with 200 μl/well of 1× blocking buffer at 37° C. for 1 hour. Then the wells were washed 3 times with 1×PBST and were incubated with 50 μl/well of antibody streptavidin-HRP diluted 1/200 at 37° C. for 1 hour. Then the wells were washed 3 times with 1×PBST and incubated with 50 μl/well of TMB/E solution for 15 min. Then the reaction result was analyzed by measuring optical density at 630 nm using an ELISA reader to confirm the biotinylation. 
       EXAMPLE 3. Measurement of Cholesterol Concentration by Immunological Method 
     3-1. Measurement of Cholesterol using ELISA 
       [0090]    The antibodies prepared in Example 1 and CDC proteins prepared in Example 2 were used to determine the concentration of cholesterol. Each of CDC proteins was allowed to bind cholesterol and anti-HDL-C antibody (#11C1) or anti-LDL-C antibody (#4C2) was used to detect HDL-C and LDL-C, respectively. 
         [0091]    Specifically, 50 μl/well of SLO, SLO_D4, PLY, PFO, LLO at the concentration of 1 μg/ml prepared in Example 2 was added to each well of 8 well ELISA strip and incubated at 37° C. for 2 hours. Then the strip was washed 3 times with 0.1% PBST and 200 μl/well of blocking buffer was added to each well followed by incubation at 37° C. for 1 hour. Then the strip was washed 3 times with PBST and incubated with HDL or LDL which was serially diluted from 2 μg/ml to 2 ng/ml at 37° C. for 1 hour. Then the strip was washed 3 times with 0.1% PBST and incubated with 50 μl/well of antibody at the concentration of 1 μg/ml prepared in Example at 37° C. for 1 hour. Then the strip was washed 3 times with 0.1% PBST and incubated with 50 μl/well of anti-goat mouse IgG HRP as a secondary antibody diluted 200 ng/ml at 37° C. for 1 hour. Then the strip was washed 3 times with 0.1% PBST and incubated with 100 μl/well of TMB/E solution for 15 min at RT. Then the reaction result was analyzed by measuring optical density at 650 nm using an ELISA reader. Results are shown in  FIG. 10  and  FIG. 11 . 
         [0092]      FIG. 10  shows the result of HDL-C concentration measured in which cholesterols were bound with CDC (SLO, SLO_D4, LLO, PFO, PLY) and detected using an antibody #11C1 which recognizes Apo A-I, main components of HDL-C. This indicates that CDCs when used as a capture are able to detect HDL-C level in a concentration dependent manner up to 30 nmg/ml. 
         [0093]      FIG. 11  shows the result of LDL-C concentration measured in which cholesterols were bound with CDC (SLO, SLO_D4, LLO, PFO, PLY) and detected using an antibody #4C2 which recognizes Apo B-100, a main components of LDL-C. This indicates that CDCs when used as a capture are able to detect LDL-C level in a concentration dependent manner up to 60 nmg/ml. 
       3-2. Measurement of Cholesterol using Rapid Kit 
       [0094]    CDC (PLY) fixed on a NC membrane as a capture and LDL antibody (#4C2) as a detection antibody were used to determine the concentration of LDL-C. 
         [0095]    Specifically, 2.6 mg/ml PLY and 1 mg/ml DNPBSA were applied at 29 mm and 34 mm NC membrane (135), respectively, as a line and the membrane was dried at 37° C. for 1 hour, which was dried again overnight in a desicator with relative humidity of 20% or lower. Then 903 sample pad and T415 absorbent pad were assembled on the membrane. 
         [0096]    Selected anti-LDL #4c2 and 0.1M sodium bicarbonate were mixed. Then FPR-648 was added at the weight ratio of Ab:fluorescent=10:1 and incubated at 4° C. overnight. Then antibody conjugated to fluorescent material was then purified using sephadex G-25. This antibody was then added to DDB to prepare DB. 
         [0097]    For a cartridge test, 1000 μl of DB was added to 1 μl of a real sample and mixed by shaking 10 times. Then 75 μl of the mixture was applied to the sample pad prepared above and incubate for 15 min. Then the result was analyzed using iChroma® (Boditech Med), in which Hitachi device (Hitachi 7020, Japan) was used as a comparative device and the same test as above was performed. The correlation with the Hitachi device was found to 0.87. The specificity and sensitivity were found to be 88% and 95%, respectively. Results are shown in  FIG. 12  and in Table 1 below. The correlation coefficient of 0.87, and the specificity and sensitivity of 88% and 95% indicate that the present method can be efficiently and conveniently used to detect LDL-C in clinic.