898 2008,22 (6) :898 903 Journal of Nuclear Agricultural Sciences :100028551 (2008) 062898206 ELISA 1,2 1 4 3 3 3 4 3 (11, 712100 ;21, 450001 ; 31, 450002 ;41, 453003) : (BSA2ENR), ELISA, ELISA (competitive ELISA ENR2Kit),: 2, IgG 1,4G12B3 ENR mab ELISA 1 :11024 10 6, ( Ka) 9 10 10 LΠmol ; ELISA 0105 10116 gπl 0105 gπl ( IC 50 ) 111 gπl, (CR %) 0101 %, 8115 % 8716 % 8413 % 95 %, ELISA ENR2Kit, 6 ELISA, ENR : ;;ELISA SCREENING OF HYBRIDOMA SECRETING MCAB TO ENROFLOXACIN AND DEVELOPMENT OF COMPETITIVE ELISA ENR2KIT ZHAO Yin2li 1,2 WANGJian2hua 1 WANG Zi2liang 4 TENG Man 3 LIU Qing2tang 3 DENG Rui2guang 3 FAN Guo2ying 4 ZHANG Gai2ping 3 (11 College of Veterinary Medicine, Northwest A &F University, Yangling, Shaanxi 712100 ; 21 College of Bioengineering, Henan University of Technology, Zhengzhou 450001 ; 31 The Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, Henan 450002 ; 41 College of Animal Sciences, Henan Institute of Science and Technology, Xinxiang, Henan 453003) Abstract :Immunogen antigen was prepared by active ester method coupling enrofloxacin to BSA. BalbΠc mice were immunized with BSA2ENR. Hybridoma lines that secreted antibody against ENR were generated with cell fusion and Screened of hybridoma was screened by indirect competitive ELISA. ENR mab was generated by inducing ascites in mice. A competitive ELISA kit for detect ENR was developed with ENR mab and its traits were tested. Two hybridoma lines were filtered and the best one was 4G1 - B3, which secreted ENR mab with indirect ELISA titers of 1 11024 10 6 in ascites. Isotype of the two mab was IgG 1. ENR mab had a high affinity constant ( Ka) with 9 10 10 LΠmol. The ENR2Kit had the linear detection range of 0105 10116 gπl, the sensitivity of 0105 gπl and a good sensitivity with an IC 50 of 111 gπl to ENR, cross2reactivity to other compounds less than 0101 %. The average recoveres of ENR spiked in chicken, liver, fish and milk were 8115 %, 8716 %,8413 % and 95 % respectively. :2008204223 :2008207224 :, (2006BAK02A21) : (19732),,,,, E2mail :zhaoyinli73 @yahoo. com. cn : (19602),,,,,E2mail :zhanggaiping2003 @yahoo. com. cn ; (19482),,,,, E2mail :jhwang1948 @sina. com ' 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
6 ELISA 899 The coefficient variation was below 10 %. The competitive ELISA ENR2Kit possesses characters of rapidity, sensitivity, specificity and briefness, which is to be used for the rapid detection of ENR residues in animal food. Key words :enrofloxacin ; hybridoma ; competitive ELISA ENR2Kit ( ENR),,,,,, 0101molΠL PBS (NaCl 8g KH 2 PO 4 012g Na 2 HPO 4 12 H 2 O 219g KCl 012g dd H 2 O 1000ml) ;( PBST) 0105 % Tween20 PBS ; (CBS) [1,2 ], 011molΠL ph916 ( Na 2 CO 3 01795g NaHCO 3 1147g dd H 2 O 500ml) ;, 5 % PBST; TMB - 30 gπkg, 10 gπkg ; 2molΠL ( HPLC) ( HPLC2MS) 11112,LEICA ;CO 2 [3,4 ],,Precision ;,,,,Beckman ;550,Bio2Rad,, 112 ( ELISA) 11211 ( ENR),,, [5,6 ] ( ENR2, BSA) 11212 6, BalbΠc,50 g 012 mlπ,,,,,3 ( ENR mab), 3 1, ELISA ( competitive ELISA ENR (pab) ELISA ENR2Kit), ELISA ENR pab ENR ( IC 50 ), 1 111 11111 BalbΠc (SPF ),, ; NS0 (BSA) (OVA) (HRP) 12232(32 ) ( EDC) N2 (NHS) ( FCA) (FIA) RPMI1640 HAT HT PEG1500 Sigma ; ( GaMIgG2HRP), ;,, 99 % ;3,3,5,5 2 (TMB) ;,AR ; IC 50 11213 7d,,,, 25 g 4 5,,,,, (5 ),,, 37, 1min 1ml PEG1500, 90s 15ml (30s 1ml,30s 3ml,30s 11ml) 37 5min,, HAT,
900 22 () 96,HAT 10 HT, 14, ELISA ELISA, 24,7d,, 1 HT 5-7d,, 24,,, 5 10 5 Πml,,14d 4 12000rpm 30min,, ENR mab ELISA ( Ka) [7,9 ] Ig 1 ELISA 11214 ENR2Kit 1121411 ( HRP2ENR) [5 ], ELISA,1 1000 ELISA 1121412 ENR mab HRP2ENR [8 ] ENR2Kit ENR mab HRP2ENR 1121413 ELISA [10 ], PBS ( ), 256 128 64 32 16 8 4 2 1 gπl, : (1) gamigg, 4 gπml,100 lπ,4,pbst, 3, 3 min ; (2) 5 % PBST,300 lπ, 37,1h,PBST ; (3),100 lπ,37,30min, PBST ; (4) (256 1 gπl), 0 ; 50 l ( HRP2 ENR), 37, 30min, PBST ; ( 5), 100 lπ,10min ; (6), 1121414 ENR mab ENR BΠB0 (B ENR A 450,B0 ENR 0 A 450 ),,, (LOD) ( IC 50 ),20,A 450 ( X) ( SD), LOD (X22s )ΠX [11 ] ENR ENR2Kit, IC 50 1121415 ENR ENR,ELISA IC 50, ENR mab ENR IC 50 IC 50 (CR %), 1121416 [12 ] :1g 1ml ( PBS = 1 1),, 5s, 30min, 4 12000rpm 1h, PBS 1 10,8 32 64 gπl ; [10 ],0101molΠL PBS 1 1 50 %,, 8 32 64 gπl 6, (CV) 1121417 PBS 5,ENR2Kit, 1121418 30 60 90 120 150 180d ENR2Kit ELISA, ( IC 50 ) 2 211 14d,,1 10 ELISA, 10 110, 10 ELISA, IC 50 10 gπl 10 24 7d 2,IC 50 2 4 gπl 2 (4G1 5D2)
6 ELISA 901 4G1 516 10 5 Πml,5D2 217 10 5 Πml HT,4G1 1112, 5D2 1135 96 7 d 70 ELISA, 70 OD 015 17,ELISA, IC 50 IC 50 1 4 gπl 3 24 7 10 d,ic 50 1 2 gπl(4g12g1 4G12B3), 4G12G1 4G12B3,, 50 %, ELISA,4G12B3 9 10 10 LΠmol,4G12G1 415 10 10 LΠmol, 1, [13,14 ], Ig IgG1 4G12B3 ELISA IC 50 111 gπl 2 ENR24G12B3 ENR Fig. 2 Inhibitory curve of 4G12B3 mab 21213 1, 0101 %, 1 4G12B3 Table 1 The cross reactivity of 4G12B3 mab inhibitors and multi antibiotics IC 50 ( gπl) cross2reactivity( %) enrofloxacin 111 100 ciprofloxacin danofloxacin difloxacin norfloxacin sulfadiazine chloramphenicol streptomycin gentamycin ampicillin 1 Fig. 1 Association constant ( Ka) curve of ENR mab 212 21211 ENR mab HRP2ENR,ENR mab 1 128000 ; HRP2 ENR 1 1000 21212 ENR mab ENR 2, Y = - 012538 X + 015095, R 2 = 019913 ELISA 20, 1102, 01085 ;LOD (B0-2s)ΠB0, LOD = 0105 gπl, ENR2Kit 0105 gπl ENR2Kit 0105 10116 gπl ENR mab ENR 21214 2, 7718 % 8615 %,8115 %,CV 514 % 9181 %, 7162 % ; 83 % 9117 %, 8716 %,CV3155 % 814 %,611 % ; 8014 % 8918 %, 8413 %, CV 513 % 10145 %,7165 % ; 9015 % 9814 %,95 %,CV 416 % 915 %, 618 % ENR2Kit 21215 3 5 IC 50 :PBS 111 gπl 1145 gπl 210 gπl 2161 gπl 2194 gπl,, IC 50, ENR2Kit,
902 22 2 ENR2Kit Table 2 Recovery test of ENR added to different samples sample chicken liver fish milk ENR amount of ENR( gπl) by ENR2Kit ( n = 6) measured value ( gπl) Recovery( %) CVΠ% 8 6192 0153 8615 6163 7166 32 2517 1139 8013 4134 514 64 49179 4188 7718 7163 9181 8 7105 0125 8811 3113 3155 32 26156 1165 83 5116 6122 64 58169 4193 9117 717 814 8 7118 0175 8918 9138 10145 32 26143 119 8216 5194 712 64 51146 2173 8014 4127 513 8 7187 0175 9814 9138 915 32 30172 1141 96 4141 416 64 57192 3165 9015 517 613 3 ELISA Fig. 3 Effect of different sample diluted solution on competitive ELISA ENR2Kit 21216 4 1 6, ELISA, IC 50 1 2 gπl, R 2 0198,, 6 3 311,,, (BSA) (OVA), BSA,OVA ELISA, [5,6,15 ], EDC NHS,, ;,,,,, 1 29, 1 16 1 10 1 5 4, 4 ELISA ELISA, 1 10, 1 10000, IC 50 10 gπl, ( ),, ELISA ELISA,,,,, 312 ELISA PEG, HAT,7d, 90 %, 15 % ELISA ELISA,,,2 4G12B3 ENR mab ELISA 1 1024000, ( Ka) 9 10 10 LΠmoL, ELISA 0105 10116 gπl, ( IC 50 ) 111 gπl, ( CR %) 0101 %,,, ;,, ;,,, ENR, PBS 2, 86 % 99 % ;,PBS(VΠV = 1 1),PBS 1 10,
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