2004 62 1 71 77 ACTA CHIMICA SINICA Vol 62 2004 No 1 71 77 ( HCG) Ξ ( 100085) Ξ 32(2 2 )242 242(3 2 ) 21 22 (AMPPD)2 (ALP) ( HCG) 13 0115 500 miu/ ml ; (C V %) (C V %) 18 % ; 80 % 116 % ; 01965 (HCG) (HCG) Micro2plate Magnetic Chemiluminescence Enzyme Immunoassay for Rapid Sensitive Determination of Human Chorionic Gonadotropin ( HCG) ZHAO Li2Xia LIN Jin2Ming Ξ QU Feng ( Research Center for Eco2environmental Sciences Chinese Academy of Sciences Beijing 100085) Abstract A micro2plate magnetic chemiluminescence enzyme immunoassay for the determination of human chorionic gonadotropin ( HCG) based on the improvement in the method of the magnetic immunoenzyme spectrophotometric assay was established The enzyme alkaline phosphatase ( ALP) was used as labeled reagent which reacted with the chemiluminescence reagent of 32(2 2spiroadamantane)242methoxy242(3 2phosphoryloxy) phenyl21 22dioxetane (AMPPD) in buffer solution The sensitivity of the present chemiluminescent method was 13 times higher than that of spectrophotometry The linear range was 0115 500 miu/ ml HCG; the within run and between run CVs were both below 18 % This method was applied to detect HCG in serum and the percent recovery was in the range of 80 % 116 % Compared with other chemiluminescence immunoassays the correlation coefficient was 01965 It was firstly reported to use enzyme immunoassay for the detection of HCG in saliva which will break a new way for establishing a non2invasive method Keywords chemiluminescence human chorionic gonadotropin ( HCG) enzyme immunoassay saliva ( HCG) HCG 36700 Da [2] [3 4] [5] HCG [6 8] DPC [9] ACS :180 [1] HCG Elecsys @ 2010 [10] HCG Ξ E2mail : jmlin @mail rcees ac cn Received May 7 2003 ; revised July 11 2003 ; accepted August 31 2003 (No 20125514)
2500 Hz 10 L 20 L 15 min 20 L 5 min 1 min - 3 (2) - Rattle [11] (FITC) L 200 ml ; HCG FITC 1/ 10[ (1) ] (3) [18] HCG 1/ 3 1 2 2 10 L 30 min LP2 [12 17] 5000 10 100 [18] [19] 1 3 HCG Deacon [20] (1) HCG HCG 1500 r/ min 10 min 32(2 2 )242 242(3 2 ) (2) 21 22 (AMPPD) [18 ] 4 1 (30 ) 3000 r/ min 10 min 13 HCG 3000 r/ min 10 min - 20 HCG 1 1 1 ALP FITC LP25000 ( MICROTEC NITION - - FITC ) 8 12 / (SUMOLIN ) 10 100 L ; ( FITC ( ) ) ( ) 2500 Hz ( PMP) ( ) ALP PMP ph = 1015 (ALP 1 U/ ml Sigma ) ; HCG ALP HCG (FITC) HCG FITC ( ) ; :011 mol/ L Tris2HCl20105 % Tween 20 ph 714 ; : 011 % ; :ph = 915 Na 2 CO 3 2NaHCO 3 ; :AMPPD (DPC ) 72 Vol 62 2004 1 2 1 2 1 (1) 96 100 200 HCG HCG 2 2 1 AMPPD ALP AMPPD ALP 7 d AMPPD2 ALP ( 1)
No 1 : (HCG) 73 1 Figure 1 Principle schematic diagram of chemiluminescence magnetic enzyme immunoassay HCG HCG 13 2 2 2 2 1 ph 915 1 ml NaHCO 3 2Na 2 CO 3 5 L (1 10-5 U/ L) AMPPD 30 30 min ( 2) AMPPD 1 10 L AMPPD 10 L AMPPD 10 L/ ml 2 2 2 ph AMPPD 10 L ph Na 2 CO 3 2 2 NaHCO 3 AMPPD2ALP Figure 2 Effect of the AMPPD concentration on CL intensity ( 3) ph : ph 915 30 min 015 h
74 Vol 62 2004 1 3 ph 1 L P/ N ; 50 L Figure 3 Effect of reaction time and ph on the CL intensity ph = 9 50 ; Ξ ph = 9 90 ; ph = 10 40 3 3 1/ 3 210 2 3 2 1 5 10 20 50 L ; HCG 10 L AMPPD AMP - D 1/ 10 015 h ph 1 ph Table 1 P/ N data in different incubation volumes p K a 910 HCG/ L 1 5 10 20 50 ph P/ N a 35 8 47 2 42 8 46 5 28 1 a ph 915 30 min 2 3 3 2 3 2 3 1 2500 Hz ( Ag) m 2 ( 5) 1 min (Ab) n [21] 96 ( P/ N) 1 h 96 P/ N 4 12 24 40 48 384 384 4 Figure 4 Effect of different dilutors of anti2hcgon the P/ N 5 Figure 5 Schematic diagram of microplate magenetic separation :
No 1 : (HCG) 75 2 4 96 / 4 3 12 3 d 515 % 1815 % ( 4) 2 80 % 116 % ; 2 7 ; 2 7 1 15 DPC x y 2 y = 11097 x - 21618 r = 01965 Table 2 Study of non2specific adsorption 7 HCG / (miu ml - 1 ) 0 1637 1552 5 1274 5893 12 5 1425 11124 25 1698 16588 2 6 5 DPC 2 5 HCG 6 (5 s ) HCG 0115 500 miu/ ml 8 % HCG : 8 7 DPC 01072 miu/ ml Figure 7 Correlation between proposed methods and the methods of ( DPC 3) 2 7 2 ( 6) 136 % 85 % 120 % 1/ 10 1/ 100 [18] 3 6 HCG Figure 6 Calibration curve of chemiluminescence signal ( 5 s integrate) vs HCG concentration 1 h 96 HCG 13
76 Vol 62 2004 3 Table 3 Sensitivity of non2isotopic immunoassays of HCG compared Assay method Label Detection limit/ (miu ml - 1 ) Solid support Ref MMCLEIA ALP 0 072 Magnetic partical (labeled anti2fitc) IEMA ALP < 1 Magnetic partical (labeled anti2fitc) [ 19 ] Immulite (DPC) ALP 0 5 ( Polystyrene) bead [ 8 ] Elecsys @ 2010 Ru(bpy) 2 + 3 Magnetic partical [ 11 ] Liaison @ 0 5 Magnetic partical ACS:180 (Bayer) AE 1 Magnetic partical [ 10 ] ALP : ;Ru(bpy) 2 3 + : ;AE: MMCLEIA : ; IEMA ; Immulite (DPC) : DPC ; Elecsys @ 2010 : ;ACS:180 (Bayer) ACS:180 4 HCG Table 4 Precision of micro2plate magnetic chemiluminescent immunoenzymetric HCG assay Sample Intra2assay ( n = 12) Inter2assay ( n = 30 detection of three days) Mean/ (miu ml - 1 ) Range/ (miu ml - 1 ) SD C V / % 1 240 4 185 8 286 4 40 6 9 9 2 155 8 112 5 181 1 28 0 10 9 3 54 2 40 0 70 3 10 0 11 5 4 18 5 15 5 22 8 2 3 5 5 1 136 4 119 2 154 8 10 5 13 7 2 232 6 212 8 252 9 15 8 18 7 3 227 3 212 8 238 9 9 0 11 5 Sample Dilutor Observed value/ (miu ml - 1 ) 1 2 5 HCG Table 5 HCG dilution results for two samples Expected value/ (miu ml - 1 ) Recovery/ % 1 1 20 2 0 70 0 60 116 3 0 32 0 40 80 4 0 28 0 30 93 1 85 80 2 48 00 42 90 112 3 31 20 28 60 109 4 22 50 21 45 105 5 17 00 17 16 99 10 8 54 8 50 101 6 HCG Table 6 Preliminary determination of HCG in the saliva Sample 1 2 Added/ (miu ml - 1 ) Determined/ (miu ml - 1 ) Recovery/ % 0 0 412 2 5 3 44 120 12 5 17 43 136 50 61 82 122 0 0 486 2 5 3 62 85 12 5 16 05 116 50 48 65 94
No 1 : (HCG) 77 Sample 3 4 5 Added/ (miu ml - 1 ) Determined/ (miu ml - 1 ) Recovery/ % 0 0 599 2 5 3 73 85 12 5 14 34 101 50 59 02 114 0 0 384 2 5 2 95 103 12 5 13 03 101 50 51 65 102 0 9 792 12 5 24 91 121 50 61 63 104 1 4 4 ; 5 30 References 1 Swaminathon N Bahl OP Fed Proc 1970 29 599 2 Xu R 2Q Zhonghua Fuchanke Zazhi 1981 16 29 ( in Chinese) ( 1981 16 29 ) 3 Kokado A ; Arakawa H ; Maeda M Anal Chim Acta 2000 407 119 4 He Z2H ; Gao N ; Jin W2R J Chromatogr B 2003 784 343 5 Nakamura N ; Lim T2K ; Jeong J 2M ; Matsunaga T Anal Chim Acta 2001 439 125 6 Zhang F ; Li Q2G ; Li Z2Q ; Chen J Chin J Anal Chem 1993 21 698 (in Chinese) ( 1993 21 698 ) 7 Vankrieken L ; Hertogh R D Clin Chem 1995 41 36 8 Lin J 2M ; Tsuji A ; Maeda M Anal Chim Acta 1997 339 139 9 Han P2Z Foreign Medicine The Fascicule of Radiomedicine and 12 Filaire E ; Bernain X ; Sagnol M ; Lac G Eur J Appl Physiol 2001 86 179 13 Morineau G ; Boudi A ; Barka A Gourmelen M ; Degeilh F ; Hardy N ; Al2Halnak A ; Soliman H ; Gosling J P ; Julien R ; Brerault J 2L ; Boudou P ; Aubert P ; Villette J 2M ; Pruna A ; Galons H ; Fiet J Clin Chem 1997 43 1397 14 Cook N J ; Scahefer A L ; Lepage P ; Jones S D M J Agric Food Chem 1997 45 395 15 Yao J 2K ; Moss H B ; Kirillova G P Clin Biochem 1998 31 187 16 Putignano P ; Dubin A ; Toja P ; Invitti C ; Bonfanti S ; Redaelli G ; Zappulli D ; Cavagnini F Eur J Endocrinol 2001 145 165 17 Anfossi L ; Tozzi C ; Giovannoli C ; Baggiani C ; Giraudi G Anal Chim Acta 2002 468 315 18 Lac G Pathol Biol 2001 49 660 19 Mao H2Y ; Zhao X Zunyi Yixueyuan Xuebao 1997 20 67 (in Chinese) ( 1997 20 67 ) 20 Deacon J K ; Thomson A M ; Page A L ; Stops J E ; Nuclearmedicine 2000 24 196 (in Chinese) Roberts P R ; Whiteley S C ; Attridge J W ; Love C ( 2000 24 A ; Robinson G A ; Davidson G P Biosen Bioelectron 196 ) 1991 6 193 10 Korest J 2C ; Masse J ; Lane A Clin Biochem 1998 21 Xie S 2W ; Yang G2Z ; Xu Y2P Clinical Immunology 31 81 Technique Shanghai Science and Technology Press Shanghai 11 Rattle S J ; Purnell D R ; Williams P I M ; Siddle 1982 p 21 (in Chinese) K ; Forrest G C Clin Chem 1984 30 1457 ( 1982 p 21) (A0305074 SHEN H ; ZHENG G C )