ACTA CHIMICA SINICA (HCG), miu/ ml ;
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1 ACTA CHIMICA SINICA Vol No ( HCG) Ξ ( ) Ξ 32(2 2 ) (3 2 ) (AMPPD)2 (ALP) ( HCG) miu/ ml ; (C V %) (C V %) 18 % ; 80 % 116 % ; (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 ) 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 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 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 Da [2] [3 4] [5] HCG [6 8] DPC [9] ACS :180 [1] HCG 2010 [10] HCG Ξ E2mail : rcees ac cn Received May ; revised July ; accepted August (No )
2 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/ L 30 min LP2 [12 17] [18] [19] 1 3 HCG Deacon [20] (1) HCG HCG 1500 r/ min 10 min 32(2 2 ) (3 2 ) (2) (AMPPD) [18 ] 4 1 (30 ) 3000 r/ min 10 min 13 HCG 3000 r/ min 10 min - 20 HCG ALP FITC LP25000 ( MICROTEC NITION - - FITC ) 8 12 / (SUMOLIN ) 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 (1) HCG HCG AMPPD ALP AMPPD ALP 7 d AMPPD2 ALP ( 1)
3 No 1 : (HCG) 73 1 Figure 1 Principle schematic diagram of chemiluminescence magnetic enzyme immunoassay HCG HCG ph ml NaHCO 3 2Na 2 CO 3 5 L ( U/ L) AMPPD min ( 2) AMPPD 1 10 L AMPPD 10 L AMPPD 10 L/ ml ph AMPPD 10 L ph Na 2 CO NaHCO 3 AMPPD2ALP Figure 2 Effect of the AMPPD concentration on CL intensity ( 3) ph : ph min 015 h
4 74 Vol 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 = / L ; HCG 10 L AMPPD AMP - D 1/ h ph 1 ph Table 1 P/ N data in different incubation volumes p K a 910 HCG/ L ph P/ N a a ph min Hz ( Ag) m 2 ( 5) 1 min (Ab) n [21] 96 ( P/ N) 1 h 96 P/ N Figure 4 Effect of different dilutors of anti2hcgon the P/ N 5 Figure 5 Schematic diagram of microplate magenetic separation :
5 No 1 : (HCG) / d 515 % 1815 % ( 4) 2 80 % 116 % ; 2 7 ; DPC x y 2 y = x r = Table 2 Study of non2specific adsorption 7 HCG / (miu ml - 1 ) DPC 2 5 HCG 6 (5 s ) HCG miu/ ml 8 % HCG : 8 7 DPC miu/ ml Figure 7 Correlation between proposed methods and the methods of ( DPC 3) ( 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
6 76 Vol Table 3 Sensitivity of non2isotopic immunoassays of HCG compared Assay method Label Detection limit/ (miu ml - 1 ) Solid support Ref MMCLEIA ALP Magnetic partical (labeled anti2fitc) IEMA ALP < 1 Magnetic partical (labeled anti2fitc) [ 19 ] Immulite (DPC) ALP 0 5 ( Polystyrene) bead [ 8 ] 2010 Ru(bpy) Magnetic partical [ 11 ] 0 5 Magnetic partical ACS:180 (Bayer) AE 1 Magnetic partical [ 10 ] ALP : ;Ru(bpy) : ;AE: MMCLEIA : ; IEMA ; Immulite (DPC) : DPC ; 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 / % Sample Dilutor Observed value/ (miu ml - 1 ) HCG Table 5 HCG dilution results for two samples Expected value/ (miu ml - 1 ) Recovery/ % HCG Table 6 Preliminary determination of HCG in the saliva Sample 1 2 Added/ (miu ml - 1 ) Determined/ (miu ml - 1 ) Recovery/ %
7 No 1 : (HCG) 77 Sample Added/ (miu ml - 1 ) Determined/ (miu ml - 1 ) Recovery/ % ; 5 30 References 1 Swaminathon N Bahl OP Fed Proc Xu R 2Q Zhonghua Fuchanke Zazhi ( in Chinese) ( ) 3 Kokado A ; Arakawa H ; Maeda M Anal Chim Acta He Z2H ; Gao N ; Jin W2R J Chromatogr B Nakamura N ; Lim T2K ; Jeong J 2M ; Matsunaga T Anal Chim Acta Zhang F ; Li Q2G ; Li Z2Q ; Chen J Chin J Anal Chem (in Chinese) ( ) 7 Vankrieken L ; Hertogh R D Clin Chem Lin J 2M ; Tsuji A ; Maeda M Anal Chim Acta Han P2Z Foreign Medicine The Fascicule of Radiomedicine and 12 Filaire E ; Bernain X ; Sagnol M ; Lac G Eur J Appl Physiol 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 Cook N J ; Scahefer A L ; Lepage P ; Jones S D M J Agric Food Chem Yao J 2K ; Moss H B ; Kirillova G P Clin Biochem Putignano P ; Dubin A ; Toja P ; Invitti C ; Bonfanti S ; Redaelli G ; Zappulli D ; Cavagnini F Eur J Endocrinol Anfossi L ; Tozzi C ; Giovannoli C ; Baggiani C ; Giraudi G Anal Chim Acta Lac G Pathol Biol Mao H2Y ; Zhao X Zunyi Yixueyuan Xuebao (in Chinese) ( ) 20 Deacon J K ; Thomson A M ; Page A L ; Stops J E ; Nuclearmedicine (in Chinese) Roberts P R ; Whiteley S C ; Attridge J W ; Love C ( A ; Robinson G A ; Davidson G P Biosen Bioelectron 196 ) Korest J 2C ; Masse J ; Lane A Clin Biochem Xie S 2W ; Yang G2Z ; Xu Y2P Clinical Immunology 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 ( 1982 p 21) (A SHEN H ; ZHENG G C )
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