1、 KSKSKSKSKSKSKSK KSKSKS KSKSK KSKS KSK KS KS P 1015 KS P 1015:2012 2012 1 2 http:/www.kats.go.krKS P 1015:2012 : ( ) ( ) () () ( ) : (http:/www.standard.go.kr) ： ：2012 1 2 2012-0002 ： ： ( 02-509-7294) (http:/www.kats.go.kr). 10 5 , . KS P 1015:2012 i ii 1 1 2 1 3 .1 4 .2 4.1 .2 4.2 .2 4.3 .2 4.4 .2
2、4.5 3 5 .3 5.1 .3 5.2 .3 5.3 3 5.4 3 6 .4 7 , .4 A( ) .5 B( ) .6 C( ) 8 D( ) 11 12 KS P 1015:2012 .13 KS P 1015:2012 ii . . , , . , , . KS P 1015:2012 Establishment of a reference material and evaluation method for troponin-based cardiovascular diagnostics 1 , . , . . . 2 . . ( ) . KS A ISO/IEC GUID
3、E 15： 2003, ISO/IEC KS A ISO GUIDE 30： 1992, KS A ISO GUIDE 33： 2005, KS P ISO 15194, KS P ISO 17511, 3 . 3.1 KS P 1015:2012 2 , , , . 3.2 1 L mL . 3.3 3.4 1 3.5 3.6 KS P ISO 17511： 2010, 3.28 4 4.1 , , . . 4.2 , , . 4.3 T, I C . . ( B ). 4.4 KS P 1015:2012 3 . I 209 C- N- 30-110 . ( C ). 4.5 . . 3
4、. . (linear epitope) (conformational epitope) . . 5 5.1 2 . ( , ) , , . 3 ( D ). 5.2 2 1 2 5 (pool) . 3 . 5.3 3 2 3 (1 ng/mL, 5 ng/mL, 30 ng/mL) . 3 4. 5.4 70 . (5.1, 5.3) . . KS P 1015:2012 4 6 5 . . 7 , 6, 7. a) ： b) ： c) ： d) ： e) ： f) ： g) , (lot), . KS P 1015:2012 5 A ( ) A.1 . KS P 1015:2012 6
5、 B ( ) B.1 (cardiac troponin complex) T (TnT), I(TnI) C(TnC) 3 . TnC TnT TnI . B.2 , cTn . . TnT TnI . cTnT 2 6 14 21 . TnI 3 14 18 5 7 . TnT 6 % 8 %, TnI 3 % 4 % , . TnT TnI T-C, T-I . B.3 I T l (rabdomyolysis) , . TnT , TnT TnT , . , . TnI 0.4 g/L , 3.1 g/L 30 . (reperfusion) . (washout) . CK TnI
6、. 4 6 100 % . 3 CK-MB , 7 14 CK-MB . T 0 6 50 % 65 % CK-MB KS P 1015:2012 7 , , 80 % . I 48 72 72 96 , 94 % . KS P 1015:2012 8 C ( ) C.1 , , . I 30-110 . C.2 Human heart cDNA library cTnI cloning 1 NIST(National Institute of Standards and Technology) SRM 2921 2010 2 . NIST, IFCC(International Federa
7、tion of Clinical Chemistry and Laboratory Medicine), AACC(American Association for Clinical Chemistry) IFCC (working group) TnI 2009 . SRM 2921 complex form 3 (TnI, TnT TnC) 1 harmonization(40 %) . TnI calibrator comparability SRM 2921 cTnI . (AMI： Acute Myocardial Infarction) cTnI complex post tran
8、slational modification(phosphorylation, oxidation ) , proteinase C-term N-term cTnI 30120 . cTnI epitope harmonization . cTnI 30120 cloning . TnI cardiac TaKaRa Human Heart cDNA library (Code No. 9504, Lot No. A206) . library pAP3neo vector(GenBank Accession No. AB003468) cloning Plasmid type . cDNA
9、 library cloning vector template (TnI-p) primer PCR . TnI-p-f： 5-CATATGACGGAGCCGCACGCCAAGTTT-3 TnI-p-r： 3-TCACTTGGTGACTTTTGCCTCTATGTCGT-5 PCR . PCR 5x PCR premix( ) 94 5 , 30 (cycles) 94 (30 ), 58 (30 ), 72 (45 ), 72 10 . DNA 1.2 % agarose gel 276 . TnI-p . T-vector(Invitrogen ) sub cloning KS P 101
10、5:2012 9 Nde1/Not1 pET28a cloning His-tagged TnI pTNIP vector . vector host BL21(DE3) pTNIP/BL21 . TnI-p (90 ) TEPHAKKKSK ISASRKLQLK TLLLQIAKQE LEREAEERRG EKGRALSTRC QPLELAGLGF AELQDLCRQL HARVDKVDEE RYDIEAKVTK C.3 24 LB/kanamycin 5 mL 37 24 . 50 L 3 mL LB/Kanamycin OD 0.40.5 induction 0.4 mM IPTG 4
11、5 4 , 3 000g/min, 10 . IPTG induction 15 % SDS-PAGE loading . C.4 LBK(kanamycin 50 g/mL) 10 mL 3 37 24 . LBK(kanamycin 50 g/mL) 400 mL 4 mL 37 2 . OD600 0.6 IPTG 1 mM 30 6 . 250 mL 6 000 xg/min, 10 , 4 . 1 g 4 mL lysis buffer(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) lysozyme 100 M 4 1 .
12、sonication 8 3 sonicator tip 8 sonication . 15 000 xg/min 30 4 . 15 % SDS-PAGE loading . Ni-NTA agarose His tagged TnI-p . Polypropylene column(Qiagen) Ni-NTA agarose(50 % slurry) 20 mL lysis buffer(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) 30 mL . TnI-p column Ni-NTA (50 mM NaH2PO4, 300
13、mM NaCl, 50 mM imidazole, pH 8.0) 40 mL column . His-tagged TnI-p (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) 20 mL Ni-NTA agarose TnI-p . 15 % SDS-polyacrylamide gel TnI-p . 5K ( 1 cm) 20 mM Tris-Cl pH 7.4, 200 mM NaCl buffer 2 L 4 3 . thrombin cleavage kit(novagen) TnI-p N-term His tag
14、. 1/10 10 thrombin cleavage/capture TnI-p 10 g diluted thrombin(1 600： 1) 1 L 37 6 15 % SDS-polyacrylamide gel His tag . His-tag TnI-p Ni-NTA agarose His peptides undigested His-tagged protein . Ni-NTA agarose column 15 % SDS-PAGE His tag TnI-p . His pore size 3K centricon(Millipore) 3 000 g 20 4 .
15、TnI-p 1 gel filtration . Resin sepadex G-75(Sigma) 1.5 cm 1 m KS P 1015:2012 10 column . (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA) 160 mL sephadex G-75 12 g column resin . . Column TnI-p 1 mL loading gel filtration . Peristaltic pump(cole-parmer ) flow speed 0.3 mL/min fraction collector(Bio-ra
16、d) fraction 3 mL . Fraction tube 15 % SDS-polyacrylamide gel . TnI-p FPLC 2 gel filtration . superode-12 column gel filtration . SDS-PAGE TnI-p 3K centricon(Millpore) 3 000g 20 4 . BCA . KS P 1015:2012 11 D ( ) D.1 1 (- P) 3 (30 ng/mL), (5 ng/mL), (1 ng/mL) Tn-I . - I (rapid diagnostic test： RDT) .
17、CE 2 (A, B) 3 . A D.1 B D.2 . 2 - P . . - P - P . . D.1 A 3 D.2 B 3 30 ng/ml( ) 5 ng/ml( ) 1 ng/ml( ) 30 ng/ml( ) 5 ng/ml( ) 1 ng/ml( ) KS P 1015:2012 12 1 EN 12287： 1999, Measurement of quantities in samples of biological origin Presentation of reference measurement procedures. 2 KS P ISO 17511, 3 NCCLS ： The reference system for the clinical Laboratory; Criteria for Development and Credentialing of Methods and Materials for Harmonization of Results; Proposed Guide
