US20200057082A1 - Method of measuring cholesterol levels by using immunogenic method - Google Patents
Method of measuring cholesterol levels by using immunogenic method Download PDFInfo
- Publication number
- US20200057082A1 US20200057082A1 US16/555,714 US201916555714A US2020057082A1 US 20200057082 A1 US20200057082 A1 US 20200057082A1 US 201916555714 A US201916555714 A US 201916555714A US 2020057082 A1 US2020057082 A1 US 2020057082A1
- Authority
- US
- United States
- Prior art keywords
- antibody
- apolipoprotein
- cdc
- cholesterol
- complex
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 title claims abstract description 163
- 238000000034 method Methods 0.000 title claims abstract description 77
- 230000002163 immunogen Effects 0.000 title 1
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 65
- 230000001419 dependent effect Effects 0.000 claims abstract description 19
- 108010056995 Perforin Proteins 0.000 claims abstract description 17
- 102000004503 Perforin Human genes 0.000 claims abstract description 17
- 102000049842 cholesterol binding protein Human genes 0.000 claims abstract description 11
- 108010011793 cholesterol binding protein Proteins 0.000 claims abstract description 11
- 102000004895 Lipoproteins Human genes 0.000 claims description 41
- 108090001030 Lipoproteins Proteins 0.000 claims description 41
- 239000000463 material Substances 0.000 claims description 33
- 108010075210 streptolysin O Proteins 0.000 claims description 28
- 101710183389 Pneumolysin Proteins 0.000 claims description 24
- 101710164436 Listeriolysin O Proteins 0.000 claims description 22
- 108010065693 Clostridium perfringens theta-toxin Proteins 0.000 claims description 19
- 238000002965 ELISA Methods 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 19
- 239000012528 membrane Substances 0.000 claims description 14
- 210000004369 blood Anatomy 0.000 claims description 13
- 239000008280 blood Substances 0.000 claims description 13
- 238000000338 in vitro Methods 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- -1 hapten Substances 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- 108010065833 botulinolysin Proteins 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- 238000003556 assay Methods 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 239000000020 Nitrocellulose Substances 0.000 claims description 4
- 108700004513 Streptococcus intermedius intermedilysin Proteins 0.000 claims description 4
- 241001134658 Streptococcus mitis Species 0.000 claims description 4
- 108010011834 Streptolysins Proteins 0.000 claims description 4
- 108010012597 alveolysin Proteins 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 229920001220 nitrocellulos Polymers 0.000 claims description 4
- 108010028874 suilysin Proteins 0.000 claims description 4
- 241000193468 Clostridium perfringens Species 0.000 claims description 3
- 241000186779 Listeria monocytogenes Species 0.000 claims description 3
- 238000003149 assay kit Methods 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 2
- 108010093201 Arcanobacterium pyogenes Pyolysin Proteins 0.000 claims description 2
- 241000193738 Bacillus anthracis Species 0.000 claims description 2
- 241000006379 Bacillus weihenstephanensis Species 0.000 claims description 2
- 241000193764 Brevibacillus brevis Species 0.000 claims description 2
- 240000001817 Cereus hexagonus Species 0.000 claims description 2
- 241000193155 Clostridium botulinum Species 0.000 claims description 2
- 241001509423 Clostridium botulinum B Species 0.000 claims description 2
- 241001509421 Clostridium botulinum C Species 0.000 claims description 2
- 241000193171 Clostridium butyricum Species 0.000 claims description 2
- 241000186581 Clostridium novyi Species 0.000 claims description 2
- 241000193449 Clostridium tetani Species 0.000 claims description 2
- 101000596238 Clostridium tetani (strain Massachusetts / E88) Tetanolysin Proteins 0.000 claims description 2
- 101710088194 Dehydrogenase Proteins 0.000 claims description 2
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 101710158696 Ivanolysin Proteins 0.000 claims description 2
- 241000186780 Listeria ivanovii Species 0.000 claims description 2
- 241000186807 Listeria seeligeri Species 0.000 claims description 2
- 241000193386 Lysinibacillus sphaericus Species 0.000 claims description 2
- 239000004677 Nylon Substances 0.000 claims description 2
- 241000178961 Paenibacillus alvei Species 0.000 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 240000004350 Prunus spinosa Species 0.000 claims description 2
- 235000010829 Prunus spinosa Nutrition 0.000 claims description 2
- 101710140587 Seeligeriolysin Proteins 0.000 claims description 2
- 241000194007 Streptococcus canis Species 0.000 claims description 2
- 241000194042 Streptococcus dysgalactiae Species 0.000 claims description 2
- 241000194046 Streptococcus intermedius Species 0.000 claims description 2
- 241000193996 Streptococcus pyogenes Species 0.000 claims description 2
- 241000194021 Streptococcus suis Species 0.000 claims description 2
- 241000186064 Trueperella pyogenes Species 0.000 claims description 2
- 229940065181 bacillus anthracis Drugs 0.000 claims description 2
- 239000011324 bead Substances 0.000 claims description 2
- 230000029918 bioluminescence Effects 0.000 claims description 2
- 238000005415 bioluminescence Methods 0.000 claims description 2
- 108010060552 cereolysin Proteins 0.000 claims description 2
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 claims description 2
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims description 2
- 239000006249 magnetic particle Substances 0.000 claims description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims description 2
- 229920001778 nylon Polymers 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 229940115920 streptococcus dysgalactiae Drugs 0.000 claims description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 2
- 238000012286 ELISA Assay Methods 0.000 claims 1
- 108010028554 LDL Cholesterol Proteins 0.000 abstract description 25
- 108010007622 LDL Lipoproteins Proteins 0.000 abstract description 23
- 102000007330 LDL Lipoproteins Human genes 0.000 abstract description 23
- 108010023302 HDL Cholesterol Proteins 0.000 abstract description 22
- 108010010234 HDL Lipoproteins Proteins 0.000 abstract description 18
- 102000015779 HDL Lipoproteins Human genes 0.000 abstract description 18
- 238000005259 measurement Methods 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 53
- 239000011347 resin Substances 0.000 description 34
- 229920005989 resin Polymers 0.000 description 34
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 32
- 239000000523 sample Substances 0.000 description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 26
- 239000000872 buffer Substances 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 17
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 16
- 238000000746 purification Methods 0.000 description 16
- 239000006228 supernatant Substances 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 13
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 13
- 229910000162 sodium phosphate Inorganic materials 0.000 description 13
- 150000001841 cholesterols Chemical class 0.000 description 12
- 210000004379 membrane Anatomy 0.000 description 12
- 235000019799 monosodium phosphate Nutrition 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 12
- 102000007592 Apolipoproteins Human genes 0.000 description 9
- 108010071619 Apolipoproteins Proteins 0.000 description 9
- 108020004705 Codon Proteins 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000010079 rubber tapping Methods 0.000 description 8
- 238000000527 sonication Methods 0.000 description 8
- 238000002372 labelling Methods 0.000 description 7
- 239000006166 lysate Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 241000282414 Homo sapiens Species 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 5
- 150000007523 nucleic acids Chemical group 0.000 description 5
- 108010031896 Cell Cycle Proteins Proteins 0.000 description 4
- 102000005483 Cell Cycle Proteins Human genes 0.000 description 4
- 102000016911 Deoxyribonucleases Human genes 0.000 description 4
- 108010053770 Deoxyribonucleases Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 102000006382 Ribonucleases Human genes 0.000 description 4
- 108010083644 Ribonucleases Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 230000000087 stabilizing effect Effects 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000005666 Apolipoprotein A-I Human genes 0.000 description 3
- 108010059886 Apolipoprotein A-I Proteins 0.000 description 3
- 102000006991 Apolipoprotein B-100 Human genes 0.000 description 3
- 108010008150 Apolipoprotein B-100 Proteins 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000192125 Firmicutes Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108700027766 Listeria monocytogenes hlyA Proteins 0.000 description 2
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000006287 biotinylation Effects 0.000 description 2
- 238000007413 biotinylation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 238000012123 point-of-care testing Methods 0.000 description 2
- 229940093430 polyethylene glycol 1500 Drugs 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000006410 Apoproteins Human genes 0.000 description 1
- 108010083590 Apoproteins Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108010004103 Chylomicrons Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010021432 Immunisation reaction Diseases 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 238000008214 LDL Cholesterol Methods 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003470 adrenal cortex hormone Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 108010067755 dinitrophenyl-bovine serum albumin Proteins 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 108010022197 lipoprotein cholesterol Proteins 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
- G01N2333/3156—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
Definitions
- the present disclosure relates to measuring the cholesterol level, particularly to measuring the cholesterol level based on immunological methods.
- 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.
- LDL Low Density Lipoprotein
- HDL High Density Lipoprotein
- LDL-C LDL cholesterol
- HDL-C HDL cholesterol
- 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.
- 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.
- CDC Choesterol Dependent Cytolysin
- anti-apolipoprotein antibodies which specifically recognize and bind to specific types of lipoprotein, which method can be advantageously used for rapid and accurate detection of cholesterols.
- 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 the cholesterol present on the surface of lipoproteins in the sample and the CDC bind to form a “lipoprotein-CDC” complex (a first complex); a step of contacting the first complex with an anti-apolipoprotein antibody by which a second complex consisting of the “lipoprotein, CDC, and antibody” is formed; and a step of detecting the second complex.
- CDC Choesterol Dependent Cytolysin
- the present disclosure provides 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 an anti-apolipoprotein antibody by which the apolipoprotein present in the lipoproteins in the sample and the antibody bind to form a “lipoprotein-antibody” complex (a third complex); a step of contacting the third complex with a CDC specifically binding to cholesterol by which a fourth complex consisting of the “lipoprotein, antibody, CDC” is formed; and a step of detecting the fourth complex.
- anti-apolipoprotein antibodies specifically recognizing apolipoproteins present in lipoproteins are used.
- Anti-apolipoprotein antibodies e.g., anti-apolipoprotein B-100 antibodies or anti-apolipoprotein A-1 antibodies
- LDL-C or HDL-C may be used depending on the type of lipoproteins in which cholesterol to be measured is present.
- various samples in need of cholesterol measurements include for example, whole blood, plasma or serum.
- CDCs are fixed onto a solid support and anti-apolipoprotein antibodies are labelled with a detectable material to detect a complex, or anti-polipoprotein antibodies are fixed onto a solid support and CDCs are labelled with a detectable material to detect a complex, without being limited thereto.
- kits to measure the cholesterol level comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and anti-polipoprotein antibodies (e.g., anti-polipoprotein B-100 antibodies or anti-polipoprotein A-1 antibodies).
- CDC Choesterol Dependent Cytolysin
- anti-polipoprotein antibodies e.g., anti-polipoprotein B-100 antibodies or anti-polipoprotein A-1 antibodies.
- CDC Choesterol Dependent Cytolysin
- cholesterol antibodies to measure the level of cholesterol.
- the present disclosure discloses a method to measure the concentration of the cholesterol using cholesterol binding proteins called CDC (Cholesterol Dependent Cytolysin) and anti-apolipoprotein antibodies.
- CDC cholesterol Dependent Cytolysin
- the present method enables a rapid and simple, simultaneous determination of LDL-C and HDL-C at a low cost and thus is applicable to various fields of art requiring their quantification, replacing the previous methods.
- FIG. 1 is a schematic diagram showing one embodiment of the present method using CDCs and anti-apolipoprotein antibodies.
- FIG. 2 is a schematic diagram showing another embodiment of the present method using CDCs and anti-apolipoprotein antibodies.
- FIG. 3A is a result of SDS-PAGE of the antibody to anti-HDL prepared in one embodiment of the present disclosure.
- FIG. 3B is a result of ELISA measuring the activity of anti-HDL antibodies identified in FIG. 3A against HDL-C according to their concentration.
- FIG. 4A is a result of SDS-PAGE of the antibody to LDL prepared in one embodiment of the present disclosure.
- FIG. 4B is a result of ELISA measuring the activity of anti-LDL antibodies identified in FIG. 4A against HDL-C according to their concentration.
- 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.
- 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.
- 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.
- 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.
- 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.
- SLO SEQ ID NO:5
- 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-1, a main component of HDL-C.
- 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.
- FIG. 12 is a graph showing the correlation of the readings generated from two different systems, in which the cholesterol concentrations were measured from human serum by a rapid kit using CDC protein and anti-LDL antibody prepared in the present disclosure using the measurement method according to one aspect of the present invention, and by a conventional device.
- the present disclosure described here is based, in part, on the discovery that the concentration of cholesterol can be determined in a sample by the use of CDC (Cholesterol Dependent Cytolysin) and antibodies against apolipoproteins present in lipoproteins.
- CDC Choesterol Dependent Cytolysin
- the cholesterols that are measured by the present methods are insoluble in an 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 can be categorized into LDL (Low Density Lipoprotein) and HDL (High Density Lipoprotein). Both LDL-C and HDL-C concentrations need to be measured in clinical settings.
- 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.
- the present invention relates to 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) binding to lipoproteins in the sample to form a first complex between “a lipoprotein and the CDC”; contacting the first complex with an anti-apolipoprotein antibody to form a second complex consisting of the “lipoprotein, CDC, and antibody”; and detecting the second complex.
- CDC Choesterol Dependent Cytolysin
- a sample by contacting a sample with CDC, cholesterol present on the surface of lipoproteins in the sample and CDC bind to each other, resulting in the formation of a first complex consisting of the lipoprotein and CDC.
- anti-apolipoprotein antibodies may specifically recognize apolipoproteins present in lipoproteins and may specifically recognize a specific type of lipoproteins.
- an anti-apolipoprotein antibody may be contacted with a first complex and as a result, bind to apolipoproteins present in the first complex, leading to the formation of a second complex consisting of the first complex and the anti-apolipoprotein antibody.
- the present invention relates to a method of measuring the cholesterol level in vitro comprising: contacting a sample in need of determination of the cholesterol level with an anti-apolipoprotein antibody specifically recognizing apolipoproteins present in lipoproteins in the sample to form a third complex between “a lipoprotein and the antibody”; contacting the third complex with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein to form a fourth complex consisting of the “lipoprotein, antibody, and CDC”; and detecting the fourth complex.
- a CDC Choesterol Dependent Cytolysin
- contacting a sample with an anti-apolipoprotein antibody allows for apolipoproteins present in lipoproteins in the sample and the anti-apolipoprotein antibody to bind to each other, resulting in the formation of a third complex consisting of the lipoprotein and anti-apolipoprotein antibody.
- contacting the third complex with CDC allows for the cholesterol present on the surface of the lipoproteins contained in the third complex and the CDC to bind to each other, resulting in the formation of a fourth complex consisting of the “lipoprotein, antibody, and CDC.”
- 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.
- ALO Anathrolysin O
- Bacillus anthracis for example UniProt Accession NO: Q81N62
- TLO Thuringiensilysin O
- B. thurigiensis for example NCBI Accession NO: YP_037419
- CLO Cereolysin O
- WLO Weihenstephanensilysin
- B. weihenstephanensis for example NCBI Accession NO: ABY46062
- LLO Literiolysin O
- LSO Seeligeriolysin O
- ILO Ivanolysin
- ivanovii for example NCBI GenBank Accession NO: AAR97343.1
- SPH Sphaericolysin
- Lysinibacillus sphaericus for example NCBI Accession NO: YP_001699692.1
- ALV Alveolysin
- Paenibacillus alvei for example Uniprot Accession NO: P23564
- BVL Brevilysin
- SLOe Streptolysin Oe
- Streptococcus dysgalactiae for example NCBI GenBank Accession NO: BAD77791
- SLO Streptolysin O
- pyogenes for example NCBI Accession NO: NP 268546.1
- SLOc Streptolysin Oc
- PSY Pseudopneumolysin
- PLY Pneumolysin
- MLY MLY
- Mitilysin from S. mitis (for example NCBI GenBank Accession NO: ABK58695)
- SLY Suilysin
- tetani for example NCBI Accession NO: NP_782466.1
- BLYb Botulinolysin B
- BLYe Botulinolysin E3
- BLYc from C. botulinum C
- NVL Novyilysin
- VLY Vaginolysin
- Gardenella vaginallis for example UniProt Accession NO: B2YGA4
- PLO Pyolysin
- Arcanobacterium pyogenes for example NCBI GenBank Accession NO: AAC45754.1
- the CDC may be LLO (Listeriolysin 0), PFO (Perfringolysin O), PLY (Pneumolysin), or SLO (Streptolysin O) or SLO-4 (Domain 4 of Streptolysin O).
- LLO Literiolysin 0
- PFO Perfringolysin O
- PLY Pneumolysin
- SLO Streptolysin O
- SLO-4 Domain 4 of Streptolysin O
- 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' binding affinity to cholesterol that is utilized in the present methods.
- various CDCs exemplified as above in addition to LLO, PFO, PLY, and SLO used herein can also be employed in the present methods.
- 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.
- 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.
- 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.
- the concentrations of each cholesterol can be measured by sequentially reacting a sample, CDC binding to cholesterol present on the surface of lipoproteins in the sample, and an anti-apolipoprotein antibody specifically binding to characteristic apolipoproteins present in lipoproteins in the sample (e.g., antibodies specific to LDL-C or HDL-C).
- an anti-apolipoprotein antibody specifically binding to characteristic apolipoproteins present in lipoproteins in the sample e.g., antibodies specific to LDL-C or HDL-C.
- the concentration of cholesterol (for example, the respective concentrations of LDL-C and HDL-C) can be measured by detecting the second or fourth complex.
- the detection of a second or a fourth complex may be achieved using various methods known in the art.
- the concentration may be determined directly by reading the signal generated from a labelling material conjugated to CDC or the anti-apolipoprotein antibody, or indirectly by reading the signal from a separate antibody labelled with chromophores, the separate antibody recognizing CDC or the anti-apolipoprotein antibody.
- Methods according to one aspect of the present invention may be embodied by the use of various methods based on immunological reactions.
- the anti-apolipoprotein antibody may be one or more of an anti-LDL antibody or an anti-HDL antibody, and particularly, may be one or more of an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody.
- the anti-apolipoprotein antibody may be two or more distinct antibodies, in which case the antibodies may be distinguishably labelled with different detectable materials.
- the distinct antibodies may be distinguishably labelled with different detectable materials.
- an anti-apolipoprotein B-100 antibody and an anti-apolipoprotein A-1 antibody which specifically bind to LDL-C and HDL-C, respectively, can simultaneously detect the levels of both LDL-C and HDL-C in a sample when they are used together while being labelled with different detectable materials.
- the antibodies which may be employed in a method according to one aspect of the present invention specifically recognize LDL-C, HDL-C, or Apo proteins contained in the above lipoproteins.
- LDL-C an antibody specific for apolipoprotein B-100 may be used
- HDL-C an antibody specific for apolipoprotein A-1.
- LDL-C an antibody specific for apolipoprotein B-100
- HDL-C an antibody specific for apolipoprotein A-1.
- these antibodies are commercially available or may be prepared by methods known in the art.
- the antibodies of interest may be prepared by the methods disclosed in the present Examples.
- the antibodies which may be used in the present disclosure include monoclonal or polyclonal antibodies.
- the antibodies which may be used in the present disclosure include full length or partial length of antibodies, aptamers, avidity multimers or peptidomimetics.
- the method according to the present invention may be performed by ELISA or a lateral flow assay.
- ELISA enzyme-linked immunosorbent assay
- ELISA is a method to quantify or detect a particular components (antigen) present in minute amount (generally below nanogram) in biological samples.
- 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.
- CDCs are fixed to a polymer (a solid support) and lipoproteins, the circulating form of cholesterol in the blood, (e.g., LDL-C or HDL-C) are applied thereto to form a complex.
- the detection uses the complex and an anti-apolipoprotein antibody recognizing a specific type of lipoproteins, which antibody may be labelled. Or additional labelled antibodies recognizing the above antibody may be used.
- an anti-apolipoprotein antibody e.g., anti-LDL antibody or anti-HDL antibody
- an antibody specific to CDCs binding to the above cholesterol is applied thereto after the unbound sample is removed, and the cholesterol level is measured using the detectable material labeling the CDC.
- one of the CDC and the anti-apolipoprotein antibody may be fixed on a solid support and the other may be labelled with a detectable material. More specifically, in one aspect of the present invention, the CDC is fixed on a solid support and the anti-apolipoprotein antibody is labelled with a detectable material.
- the CDC or the anti-apolipoprotein antibody may be fixed on a solid support in advance of contacting it with other substances (for example, a sample or complex).
- the solid support may have an inert surface to which CDCs or anti-apolipoprotein 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.
- CDCs or anti-apolipoprotein 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 but are not limited to, for example, a fluorescent moiety, a chemiluminescent moiety, a bioluminescence moiety, a magnetic particle, an enzyme, a substrate, a radioisotope and a chromophore.
- CDCs or anti-apolipoprotein antibodies 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.
- the quantitative or qualitative detection may be performed by using antibodies conjugated with an 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 e.g., HDL-C or LDL-C
- the component of interest e.g., HDL-C or LDL-C
- the quantitative or qualitative determination of the component of interest can then be determined from the amounts of anti-apolipoprotein antibodies or CDCs bound to a detectable material, which is measured by the intensity of the light or color generated.
- a detectable material e.g., Biochemistry, 2nd edition, B. D. Hames and N. M. Hooper, Springer-Verlag New York 2000, pages 112-114 may be referred for further details.
- the present invention may relate to a method of measuring the cholesterol level in vitro, the method comprising: contacting a sample in need of determination of the cholesterol level with an anti-apolipoprotein antibody to form the third complex; contacting the third complex with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein to form the fourth complex; and detecting the fourth complex using the antibody, wherein the CDC is fixed on a solid support in advance of contacting the sample with the anti-apolipoprotein antibody, the antibody is an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody, and the antibody is labelled with a detectable material.
- CDC Choesterol Dependent Cytolysin
- the present invention may relate to a kit for measuring the cholesterol level comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and an anti-apolipoprotein antibody.
- CDC Choesterol Dependent Cytolysin
- the kit may be a lateral flow assay kit.
- the kit according to one aspect of the present invention may be to measure the cholesterol level by the measurement method according to one aspect of the present invention.
- a rapid kit based on lateral flow assay may be used.
- 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 an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody is contacted with the membrane by applying the mixture at one end of the membrane.
- POCT Point of care test
- 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 can be determined by detecting the signals generated from the labeling materials conjugated to the anti-apolipoprotein antibodies bound to the apolipoproteins of lipoproteins.
- the present invention relates to a lateral flow assay kit for measuring the cholesterol level
- CDC Choesterol Dependent Cytolysin
- the CDC is fixed on a solid support, and the antibody is an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody and is labelled with a detectable material.
- mice of 6-8 weeks Female BALB/c mice of 6-8 weeks were used.
- Human HDL and LDL were purchased from ProSpec (USA), complete Freund's adjuvant and Freund's incomplete adjuvant were purchased from MP Biomedicals Inc (USA).
- Fetal bovine serum (FBS) and Dulbecco'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
- the medium contained 10% FBS.
- Pristane (2,6,10,14-tetrametheylepentadecane) and Protein G Immobilized on agarose were purchased from Sigma (USA).
- 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&D systems (USA).
- the blocking buffer were prepared by adding 1% BDA in 0.1% PBS/T.
- NC membrane HFB13504 from Millipore was used.
- Whatman 903 was used.
- 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% PB ST pH 7.4.
- DB was prepared by adding 1 ⁇ g/ml anti-LDL #4c2, and 50 ng/ml anti-DNP #3A6G9 to DDB.
- the LDLs were intraperitoneally (I.P.) injected into the mice.
- I.P. intraperitoneally
- 200 ⁇ g/200 ⁇ l of human LDL and HDL were mixed with 100 ⁇ l of complete Freund's adjuvant, and the mixture were intraperitoneally injected into BALB/c mice at 300 ⁇ l/mouse.
- the mixture of HDL, LDL and Freund's incomplete adjuvant was injected every 10 days for a total of 3-4 times.
- 500 ⁇ g/300 ⁇ l of HDL and LDL without the adjuvant was intraperitoneally injected.
- 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 ⁇ l/well and incubated overnight at 37° C. CO 2 incubator.
- 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.
- 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 ⁇ l/well and incubated in a 37° C. CO 2 incubator.
- 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 ⁇ l/well and incubated at 37° C. for 1 hour. Then the wells were washed with 0.1% PBST 3 times and 100 ⁇ l/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.
- 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.
- Homogenous clones of the cells producing antibody were isolated through cloning process as described below.
- the feeder cells were seeded onto each well of 96 well plate and incubated overnight in a 37° C., CO 2 incubator.
- the parent cells were counted and diluted to include one cell in 140 ⁇ l, which was added to each well and incubated in a 37° C., CO 2 incubator to obtain a colony.
- 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.
- BALB/C was injected with pristine at 300 ⁇ l/mouse to disable the immune system. After 7 days, the fused cells were diluted in incomplete DMEM at the concentration of 1 ⁇ 10 ⁇ circumflex over ( ) ⁇ 6, which were then intraperitoneally injected into mice at 300 ⁇ l/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.
- 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.
- 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.
- the plate was washed 3 times with 0.1% PBST and incubated with 50 ⁇ l/well of the 1 st antibody prepared by two-fold serial dilution starting from 1 ⁇ g/ml at 37° C. for 1 hour. Then the plate was washed 3 times with 0.1% PBST and incubated with 50 ⁇ l/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 ⁇ l/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.
- Pneumolysin 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).
- pET-21a.ply_BL21TM DE3 star cells were grown in 0/N and subcultured in 4.2 L LB medium containing Amp+ cam at 37° C. with shaking at 200 rpm. When O.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 column in upside down position.
- native purification buffer containing protease, DNase and RNase.
- lysates 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.
- Perfringolysin 0 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).
- pET21a Novagen
- pET-21a.pfo_BL-21 DE3 star cells were grown in 0/N and subcultured in 5.4 L LB medium containing Kan+ cam at 37° C. with shaking at 200 rpm. When the O.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 column in upside down position. The resin was stabilized before the supernatant was decanted.
- native purification buffer containing protease, DNase and RNase.
- 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 50Mm NaH2PO4, pH 8.0; 0.5 M NaCl, 20 mM imidazole
- 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. 6 , recombinant Perfringolysin 0 was successfully expressed, and purified under a native condition.
- Native Elution Buffer 50 mM NaH2PO4, pH 8.0; 0.5 M NaCl, 250 mM imidazole
- centricon ultra-filtration 10,000 MWCO
- Listeriolysin O 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).
- 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.
- native purification buffer containing protease, DNase and RNase.
- the column was eluted with 10 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).
- Native Elution Buffer 50 mM NaH2PO4, pH 8.0; 0.5 M NaCl, 250 mM imidazole
- centricon ultra-filtration 10,000 MWCO
- SLO consists of 4 domains and the domain 4 at N-terminus contains SH— group that binds to cholesterol.
- 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.
- 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.
- pET100.SLO D4-BL-21 star cells were grown in 0/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.
- 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 (50Mm 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.
- Native Wash Buffer 50Mm NaH2PO4, pH 8.0; 0.5 M NaCl, 20 mM imidazole
- 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 (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.
- Native Elution Buffer 50 mM NaH2PO4, pH 8.0; 0.5 M NaCl, 250 mM imidazole
- 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.
- Protein and biotin were mixed at the concentration ratio of 10:1 (100 ⁇ l) and 1M sodium bicarbonate was added to the mixture at 1/10 of the total volume (10 ⁇ l), which was then incubated overnight at 4° C.
- 1M sodium bicarbonate was added to the mixture at 1/10 of the total volume (10 ⁇ l), which was then incubated overnight at 4° C.
- SLO-biotin was prepared by two-fold serial dilution starting from 2 ⁇ g/ml and added to each well of 8 well strip at 5 ⁇ l/well. The wells were then incubated at 37° C. for 2 hours and washed 3 times with 1 ⁇ PBST followed by blocking with 200 ⁇ l/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 ⁇ l/well of antibody streptavidin-HRP diluted 1/200 at 37° C. for 1 hour.
- Example 1 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 present on the surface of lipoproteins and anti-HDL-C antibody (#11C1) or anti-LDL-C antibody (#4C2) was used to detect HDL-C and LDL-C, respectively.
- the strip was washed 3 times with 0.1% PBST and incubated with 50 ⁇ l/well of antibody at the concentration of 1 ⁇ g/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 ⁇ l/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 ⁇ l/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 .
- FIG. 10 shows the result of HDL-C concentration measured in which cholesterols present on the surface of lipoproteins were bound to CDC (SLO, SLO_D4, LLO, PFO, PLY) and detected using an antibody #11C1 which recognizes Apo A-1, the main component 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 ng/ml.
- FIG. 11 shows the result of LDL-C concentration measured in which cholesterols present on the surface of lipoproteins were bound to CDC (SLO, SLO_D4, LLO, PFO, PLY) and detected using an antibody #4C2 which recognizes Apo B-100, the main component of LDL-C.
- SLO SLO
- SLO_D4 LLO
- PFO PLY
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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.
Description
- The present application is a divisional application of U.S. patent application Ser. No. 15/545,656, filed Jul. 21, 2017, which was 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
- The Sequence Listing submitted in text format (.txt) filed on Aug. 29, 2019, named “SequenceListing.txt”, created on Aug. 28, 2019 (8.83 KB), is incorporated herein by reference.
- The present disclosure relates to measuring the cholesterol level, particularly to measuring the cholesterol level based on immunological methods.
- 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.
- Both LDL cholesterol (“LDL-C”) and HDL cholesterol (“HDL-C”) 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. For example, high levels of LDL-C may generate thrombus, resulting in atherosclerosis which may lead to a heart attack, and are implicated in other problems including 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, SG Thompson. By how much and how quickly does reduction in serum cholesterol concentration lower risk of ischaemic heart disease, In BMJ, 1994.).
- 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.
- One of the methods to measure the LDL-C level is based on a method called, Friedewald: LDL-c=TC-[HDL-c+TG/k], k=5). However, this equation to calculate the LDL-C level has been reported to be inaccurate when the triglyceride level is over 400 mg/dL or lipoproteins called chylomicrons are present abundantly in the blood (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). Other problems include high cost and time.
- 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.
- Therefore there exist needs to develop a more convenient and effective method to measure the concentration at a low cost.
- In the present disclosure, there is provided a method to measure the levels of LDL-C and HDL-C based on CDC (Cholesterol Dependent Cytolysin), which specifically recognizes and binds to cholesterol, and anti-apolipoprotein antibodies, which specifically recognize and bind to specific types of lipoprotein, which method can be advantageously used for rapid and accurate detection of cholesterols.
- 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 the cholesterol present on the surface of lipoproteins in the sample and the CDC bind to form a “lipoprotein-CDC” complex (a first complex); a step of contacting the first complex with an anti-apolipoprotein antibody by which a second complex consisting of the “lipoprotein, CDC, and antibody” is formed; and a step of detecting the second complex.
- Alternatively, the present disclosure provides 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 an anti-apolipoprotein antibody by which the apolipoprotein present in the lipoproteins in the sample and the antibody bind to form a “lipoprotein-antibody” complex (a third complex); a step of contacting the third complex with a CDC specifically binding to cholesterol by which a fourth complex consisting of the “lipoprotein, antibody, CDC” is formed; and a step of detecting the fourth complex.
- In the foregoing methods, various CDCs having affinity to cholesterols from gram positive bacteria may be employed.
- Still in the foregoing methods, in addition to CDCs, anti-apolipoprotein antibodies specifically recognizing apolipoproteins present in lipoproteins are used. Anti-apolipoprotein antibodies (e.g., anti-apolipoprotein B-100 antibodies or anti-apolipoprotein A-1 antibodies) from various types and/or origins specifically recognizing LDL-C or HDL-C may be used depending on the type of lipoproteins in which cholesterol to be measured is present.
- Still in the foregoing methods, various samples in need of cholesterol measurements include for example, whole blood, plasma or serum.
- 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-apolipoprotein antibodies are labelled with a detectable material to detect a complex, or anti-polipoprotein antibodies are fixed onto a solid support and CDCs are labelled with a detectable material to detect a complex, without being limited thereto.
- In another aspect, there is provided a kit to measure the cholesterol level comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and anti-polipoprotein antibodies (e.g., anti-polipoprotein B-100 antibodies or anti-polipoprotein A-1 antibodies).
- In another aspect, there is provided a use of CDC (Cholesterol Dependent Cytolysin) and cholesterol antibodies to measure the level of cholesterol.
- The present disclosure discloses a method to measure the concentration of the cholesterol using cholesterol binding proteins called CDC (Cholesterol Dependent Cytolysin) and anti-apolipoprotein antibodies. The present method enables a rapid and simple, simultaneous determination of LDL-C and HDL-C at a low cost and thus is applicable to various fields of art requiring their quantification, replacing the previous methods.
-
FIG. 1 is a schematic diagram showing one embodiment of the present method using CDCs and anti-apolipoprotein antibodies. -
FIG. 2 is a schematic diagram showing another embodiment of the present method using CDCs and anti-apolipoprotein antibodies. -
FIG. 3A is a result of SDS-PAGE of the antibody to anti-HDL prepared in one embodiment of the present disclosure. -
FIG. 3B is a result of ELISA measuring the activity of anti-HDL antibodies identified inFIG. 3A against HDL-C according to their concentration. -
FIG. 4A is a result of SDS-PAGE of the antibody to LDL prepared in one embodiment of the present disclosure. -
FIG. 4B is a result of ELISA measuring the activity of anti-LDL antibodies identified inFIG. 4A against HDL-C according to their concentration. -
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. -
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. -
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. -
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. -
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. -
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-1, a main component of HDL-C. -
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. -
FIG. 12 is a graph showing the correlation of the readings generated from two different systems, in which the cholesterol concentrations were measured from human serum by a rapid kit using CDC protein and anti-LDL antibody prepared in the present disclosure using the measurement method according to one aspect of the present invention, and by a conventional device. - The present disclosure described here is based, in part, on the discovery that the concentration of cholesterol can be determined in a sample by the use of CDC (Cholesterol Dependent Cytolysin) and antibodies against apolipoproteins present in lipoproteins.
- The cholesterols that are measured by the present methods are insoluble in an 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 can be categorized into LDL (Low Density Lipoprotein) and HDL (High Density Lipoprotein). Both LDL-C and HDL-C concentrations need to be measured in clinical settings.
- 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.
- In the present discourse, methods to determine the concentration of cholesterol in a sample have been developed by unexpected efforts.
- In one aspect, the present invention relates to 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) binding to lipoproteins in the sample to form a first complex between “a lipoprotein and the CDC”; contacting the first complex with an anti-apolipoprotein antibody to form a second complex consisting of the “lipoprotein, CDC, and antibody”; and detecting the second complex.
- In one aspect of the present invention, by contacting a sample with CDC, cholesterol present on the surface of lipoproteins in the sample and CDC bind to each other, resulting in the formation of a first complex consisting of the lipoprotein and CDC.
- In one aspect of the present invention, anti-apolipoprotein antibodies may specifically recognize apolipoproteins present in lipoproteins and may specifically recognize a specific type of lipoproteins. In one aspect of the present invention, an anti-apolipoprotein antibody may be contacted with a first complex and as a result, bind to apolipoproteins present in the first complex, leading to the formation of a second complex consisting of the first complex and the anti-apolipoprotein antibody.
- In another aspect, the present invention relates to a method of measuring the cholesterol level in vitro comprising: contacting a sample in need of determination of the cholesterol level with an anti-apolipoprotein antibody specifically recognizing apolipoproteins present in lipoproteins in the sample to form a third complex between “a lipoprotein and the antibody”; contacting the third complex with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein to form a fourth complex consisting of the “lipoprotein, antibody, and CDC”; and detecting the fourth complex.
- In one aspect of the present invention, contacting a sample with an anti-apolipoprotein antibody allows for apolipoproteins present in lipoproteins in the sample and the anti-apolipoprotein antibody to bind to each other, resulting in the formation of a third complex consisting of the lipoprotein and anti-apolipoprotein antibody.
- In one aspect of the present invention, contacting the third complex with CDC allows for the cholesterol present on the surface of the lipoproteins contained in the third complex and the CDC to bind to each other, resulting in the formation of a fourth complex consisting of the “lipoprotein, antibody, and CDC.”
- In one aspect of the present invention, 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: AB021366.1); 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 Accession NO: B2YGA4), PLO (Pyolysin) from Arcanobacterium pyogenes (for example NCBI GenBank Accession NO: AAC45754.1).
- In particular, in one aspect of the present invention, the CDC may be LLO (Listeriolysin 0), PFO (Perfringolysin O), PLY (Pneumolysin), or SLO (Streptolysin O) or SLO-4 (
Domain 4 of Streptolysin O). - 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' 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.
- 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.
- 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.
- 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.
- In the present methods, the concentrations of each cholesterol can be measured by sequentially reacting a sample, CDC binding to cholesterol present on the surface of lipoproteins in the sample, and an anti-apolipoprotein antibody specifically binding to characteristic apolipoproteins present in lipoproteins in the sample (e.g., antibodies specific to LDL-C or HDL-C).
- In a method of measuring the cholesterol level in vitro according to one aspect of the present invention, the concentration of cholesterol (for example, the respective concentrations of LDL-C and HDL-C) can be measured by detecting the second or fourth complex.
- In one aspect of the present invention, the detection of a second or a fourth 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 CDC or the anti-apolipoprotein antibody, or indirectly by reading the signal from a separate antibody labelled with chromophores, the separate antibody recognizing CDC or the anti-apolipoprotein antibody.
- Methods according to one aspect of the present invention may be embodied by the use of various methods based on immunological reactions.
- In one aspect of the present invention, the anti-apolipoprotein antibody may be one or more of an anti-LDL antibody or an anti-HDL antibody, and particularly, may be one or more of an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody.
- In one aspect of the present invention, the anti-apolipoprotein antibody may be two or more distinct antibodies, in which case the antibodies may be distinguishably labelled with different detectable materials.
- In one aspect of the present invention, when two or more anti-apolipoprotein antibodies are used, for example, when both an anti-apolipoprotein B-100 antibody and an anti-apolipoprotein A-1 antibody are used, the distinct antibodies may be distinguishably labelled with different detectable materials. Specifically, an anti-apolipoprotein B-100 antibody and an anti-apolipoprotein A-1 antibody, which specifically bind to LDL-C and HDL-C, respectively, can simultaneously detect the levels of both LDL-C and HDL-C in a sample when they are used together while being labelled with different detectable materials.
- The antibodies which may be employed in a method according to one aspect of the present invention specifically recognize LDL-C, HDL-C, or Apo proteins contained in the above lipoproteins. For example, in the case of LDL-C, an antibody specific for apolipoprotein B-100 may be used, and in the case of HDL-C, an antibody specific for apolipoprotein A-1. 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.
- In one aspect of the present invention, the method according to the present invention may be performed by ELISA or a lateral flow assay.
- In one embodiment of the present disclosure, ELISA (enzyme-linked immunosorbent assay) may be 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 (a solid support) and lipoproteins, the circulating form of cholesterol in the blood, (e.g., LDL-C or HDL-C) are applied thereto to form a complex. The detection uses the complex and an anti-apolipoprotein antibody recognizing a specific type of lipoproteins, which antibody may be labelled. Or additional labelled antibodies recognizing the above antibody may be used.
- In another example, an anti-apolipoprotein antibody (e.g., anti-LDL antibody or anti-HDL antibody) may be fixed to an inert polymer support and the biological sample is applied thereto. Then an antibody specific to CDCs binding to the above cholesterol is applied thereto after the unbound sample is removed, and the cholesterol level is measured using the detectable material labeling the CDC.
- Specifically, in one aspect of the present invention, one of the CDC and the anti-apolipoprotein antibody may be fixed on a solid support and the other may be labelled with a detectable material. More specifically, in one aspect of the present invention, the CDC is fixed on a solid support and the anti-apolipoprotein antibody is labelled with a detectable material.
- In one aspect of the present invention, the CDC or the anti-apolipoprotein antibody may be fixed on a solid support in advance of contacting it with other substances (for example, a sample or complex).
- In one aspect of the present invention, the solid support may have an inert surface to which CDCs or anti-apolipoprotein 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.
- CDCs or anti-apolipoprotein 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 but are not limited to, for example, a fluorescent moiety, a chemiluminescent moiety, a bioluminescence moiety, a magnetic particle, an enzyme, a substrate, a radioisotope and a chromophore.
- Specifically, in one aspect of the present invention, CDCs or anti-apolipoprotein antibodies may be labelled with labeling materials enabling direct or indirect detection such as, for example radioisotopes such as 3H or 125I, 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 an 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 (e.g., HDL-C or LDL-C) in the sample can then be determined from the amounts of anti-apolipoprotein antibodies or CDCs bound to a detectable material, which is measured by the intensity of the light or color generated. 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.
- In one aspect, the present invention may relate to a method of measuring the cholesterol level in vitro, the method comprising: contacting a sample in need of determination of the cholesterol level with an anti-apolipoprotein antibody to form the third complex; contacting the third complex with a CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein to form the fourth complex; and detecting the fourth complex using the antibody, wherein the CDC is fixed on a solid support in advance of contacting the sample with the anti-apolipoprotein antibody, the antibody is an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody, and the antibody is labelled with a detectable material.
- In one aspect, the present invention may relate to a kit for measuring the cholesterol level comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and an anti-apolipoprotein antibody.
- In one aspect of the present invention, the kit may be a lateral flow assay kit.
- The kit according to one aspect of the present invention may be to measure the cholesterol level by the measurement method according to one aspect of the present invention.
- In other embodiment of the present disclosure, a rapid kit based on lateral flow assay may be 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 an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody 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 can be determined by detecting the signals generated from the labeling materials conjugated to the anti-apolipoprotein antibodies bound to the apolipoproteins of lipoproteins.
- In one aspect, the present invention relates to a lateral flow assay kit for measuring the cholesterol level comprising CDC (Cholesterol Dependent Cytolysin) as a cholesterol binding protein and an anti-apolipoprotein antibody, wherein the CDC is fixed on a solid support, and the antibody is an anti-apolipoprotein B-100 antibody or an anti-apolipoprotein A-1 antibody and is labelled with a detectable material.
- 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.
- 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.
- Female BALB/c mice of 6-8 weeks were used. Human HDL and LDL were purchased from ProSpec (USA), complete Freund's adjuvant and Freund's incomplete adjuvant were purchased from MP Biomedicals Inc (USA). Fetal bovine serum (FBS) and Dulbecco'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).
- For isotyping, Pierce™ Rapid Mouse Antibody Isotyping Kits (Quickly determine mouse monoclonal antibody (MAb) class and subclass identity.) were used.
- 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&D systems (USA). The blocking buffer were prepared by adding 1% BDA in 0.1% PBS/T.
- 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% PB ST pH 7.4. DB was prepared by adding 1 μg/ml anti-LDL #4c2, and 50 ng/ml anti-DNP #3A6G9 to DDB.
- 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'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'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.
- One day before fusing cells, BALB/c mice of 7-8 weeks of age were sacrificed under CO2 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. CO2 incubator.
- 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.
- Then SP2/O cells were used as myeloid cells, which were cultured in 75T cm2 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. CO2 incubator.
- 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. 1 μg/ml of HDL, LDL was added at 50 μl/well and incubated for 2 hours at 37° C. Then each well was washed with 1×
PBST 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. CO2 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, 3rd and 4th screenings were performed and the screened cells were transferred to 75T cm2 flask. The cells were sub-cultured and the parent cells were stored frozen. - 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., CO2 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., CO2 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 cm2 T flask and finally to 75 cm2 T flask.
- 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{circumflex over ( )}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 CO2 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.
- 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.
- To remove glycine, a dialysis in 1×PBS pH 7.4 was performed. The purified antibody was confirmed to consist of a heavy chain 50-55 kDa and a light chain 20-25 kDa 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 1st antibody prepared by two-fold serial dilution starting from 1 μg/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. - 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).
- 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 0/N and subcultured in 4.2 L LB medium containing Amp+ cam at 37° C. with shaking at 200 rpm. When O.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 column 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 column in upside down position. After stabilizing the resin, the supernatants were decanted. The process was repeated one more time. - 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.
- 30 ml Native Wash Buffer (50Mm 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. - Perfringolysin 0 (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).
- 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 0/N and subcultured in 5.4 L LB medium containing Kan+ cam at 37° C. with shaking at 200 rpm. When the O.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 column 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 column in upside down position. After stabilizing the resin, the supernatants were decanted. The process was repeated one more time. - 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 (50Mm 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. 6 , recombinant Perfringolysin 0 was successfully expressed, and purified under a native condition. - 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).
- 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 bysonication 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 NaH2PO4, 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. - 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 (50Mm 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 10 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. 7 , recombinant Listeriolysin 0 (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. - 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. - 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. - pET100.SLO D4-BL-21 star cells were grown in 0/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. 10 ml 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 column 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 column in upside down position. After stabilizing the resin, the supernatant was decanted. The process was repeated one more time. - 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 (50Mm 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 (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. - 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 inFIG. 9 . - 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 volume (10 μl), which was then incubated overnight at 4° C. To pack 1 m 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.
- 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.
- 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 present on the surface of lipoproteins and anti-HDL-C antibody (#11C1) or anti-LDL-C antibody (#4C2) was used to detect HDL-C and LDL-C, respectively.
- 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 andFIG. 11 . -
FIG. 10 shows the result of HDL-C concentration measured in which cholesterols present on the surface of lipoproteins were bound to CDC (SLO, SLO_D4, LLO, PFO, PLY) and detected using an antibody #11C1 which recognizes Apo A-1, the main component 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 ng/ml. -
FIG. 11 shows the result of LDL-C concentration measured in which cholesterols present on the surface of lipoproteins were bound to CDC (SLO, SLO_D4, LLO, PFO, PLY) and detected using an antibody #4C2 which recognizes Apo B-100, the main component 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 ng/ml. - 3-2. Measurement of cholesterol using Rapid kit
- 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 in human serum (n=100).
- 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.
- 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.
- For a cartridge test, 1 μl of a human serum was added to 1000 μl of DB and mixed by shaking 10 times. Then 75 μl of the mixture was applied to the sample pad prepared above and incubated 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. -
TABLE 1 Hitachi Specificity Sensitivity type Result Positive Negative (%) (%) i-chroma Positive 15 4 88 95 Negative 2 79 Sum 100
Claims (19)
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 3H or 125I, 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.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/555,714 US20200057082A1 (en) | 2015-01-23 | 2019-08-29 | Method of measuring cholesterol levels by using immunogenic method |
US18/519,998 US20240094228A1 (en) | 2015-01-23 | 2023-11-27 | Method of measuring cholesterol levels by using immunogenic method |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2015-0011355 | 2015-01-23 | ||
KR20150011355 | 2015-01-23 | ||
KR10-2016-0007850 | 2016-01-22 | ||
KR1020160007850A KR20160091276A (en) | 2015-01-23 | 2016-01-22 | Method of measuring cholesterol level using CDC protein and antibody against cholesterol |
PCT/KR2016/000689 WO2016117956A1 (en) | 2015-01-23 | 2016-01-22 | Method for measuring cholesterol by using cdc protein and cholesterol antibody |
US201715545656A | 2017-07-21 | 2017-07-21 | |
US16/555,714 US20200057082A1 (en) | 2015-01-23 | 2019-08-29 | Method of measuring cholesterol levels by using immunogenic method |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2016/000689 Division WO2016117956A1 (en) | 2015-01-23 | 2016-01-22 | Method for measuring cholesterol by using cdc protein and cholesterol antibody |
US15/545,656 Division US10401371B2 (en) | 2015-01-23 | 2016-01-22 | Method of measuring cholesterol levels by using immunogenic method |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US18/519,998 Division US20240094228A1 (en) | 2015-01-23 | 2023-11-27 | Method of measuring cholesterol levels by using immunogenic method |
Publications (1)
Publication Number | Publication Date |
---|---|
US20200057082A1 true US20200057082A1 (en) | 2020-02-20 |
Family
ID=56708322
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/545,656 Active US10401371B2 (en) | 2015-01-23 | 2016-01-22 | Method of measuring cholesterol levels by using immunogenic method |
US16/555,714 Abandoned US20200057082A1 (en) | 2015-01-23 | 2019-08-29 | Method of measuring cholesterol levels by using immunogenic method |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/545,656 Active US10401371B2 (en) | 2015-01-23 | 2016-01-22 | Method of measuring cholesterol levels by using immunogenic method |
Country Status (2)
Country | Link |
---|---|
US (2) | US10401371B2 (en) |
KR (2) | KR20160091276A (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT201900000651A1 (en) | 2019-01-16 | 2019-04-16 | Pastore Lucio | Gene transfer technology |
KR102258048B1 (en) | 2019-05-30 | 2021-05-31 | 주식회사 캔티스 | Method for detecting amyloid beta oligomer |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140228239A1 (en) * | 2013-02-08 | 2014-08-14 | Bio-Rad Laboratories, Inc. | Affinity-based partition assay for detection of target molecules |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU762931B2 (en) | 1999-03-16 | 2003-07-10 | Serex, Inc. | Method and device for detection of Apo A, Apo B and the ratio thereof in saliva |
JP2000325097A (en) | 1999-05-21 | 2000-11-28 | Showa Denko Kk | Method and reagent for measuring lipoprotein cholesterol |
CN100516878C (en) | 2001-07-12 | 2009-07-22 | 协和美帝克斯股份有限公司 | Method of quantifying denatured lipoprotein, and quantifying reagent thereof |
JP4647927B2 (en) | 2004-03-31 | 2011-03-09 | デンカ生研株式会社 | Multiple determination of cholesterol in low density lipoprotein |
DK2208794T3 (en) | 2007-10-10 | 2016-06-06 | Denka Seiken Kk | A method and kit for the quantification of small, closely-LDL cholesterol |
-
2016
- 2016-01-22 KR KR1020160007850A patent/KR20160091276A/en not_active Application Discontinuation
- 2016-01-22 US US15/545,656 patent/US10401371B2/en active Active
-
2018
- 2018-03-23 KR KR1020180034058A patent/KR101971224B1/en active IP Right Grant
-
2019
- 2019-08-29 US US16/555,714 patent/US20200057082A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140228239A1 (en) * | 2013-02-08 | 2014-08-14 | Bio-Rad Laboratories, Inc. | Affinity-based partition assay for detection of target molecules |
Also Published As
Publication number | Publication date |
---|---|
US10401371B2 (en) | 2019-09-03 |
KR20160091276A (en) | 2016-08-02 |
US20180017587A1 (en) | 2018-01-18 |
KR20180035756A (en) | 2018-04-06 |
KR101971224B1 (en) | 2019-04-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11372001B2 (en) | Anti-human IgG4 monoclonal antibody and methods of making and using same | |
US20240094228A1 (en) | Method of measuring cholesterol levels by using immunogenic method | |
US20200057082A1 (en) | Method of measuring cholesterol levels by using immunogenic method | |
US20230151119A1 (en) | Antibodies to Symmetrically Dimethylated Arginine Analytes and Use Thereof | |
EP2535715B1 (en) | METHOD FOR MEASURING IMMUNITY OF COMPLEX OF Ku86 AND AUTOANTIBODY THEREOF, KIT USED THEREFOR, AND METHOD FOR DETERMINING CANCER USING SAME | |
KR101338517B1 (en) | Human liver carboxylesterase 1-specific indicating monoclonal antibody, hybridoma cell line producing the same and use thereof | |
EP3531129B1 (en) | Immunoassay method using anti-human bnp fragment (4-32) antibody | |
JP6037043B2 (en) | Protein quantification method specific to TRACP-5b (tartrate-resistant acid phosphatase 5b) | |
JP7475584B2 (en) | Periostin bound to immunoglobulin A, antibody that binds to periostin bound to immunoglobulin A, method for measuring periostin, reagent for measuring periostin, and method for improving accuracy of periostin measurement | |
US20160370382A1 (en) | Method for immunoassay of autoantibody against ku86, kit for use in same, and method for determination of primary hepatocellular carcinoma using same | |
JP6099393B2 (en) | Autotaxin isoform-specific antibody and detection method | |
US9017959B2 (en) | 5.9 kDa peptide immunoassay method | |
JP2022142169A (en) | Parathyroid hormone measurement method | |
US20180209967A1 (en) | Compositions and methods for detecting loa loa |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE AFTER FINAL ACTION FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: ADVISORY ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |