, 6 ELISA, SCREENING OF HYBRIDOMA SECRETING MCAB TO ENROFLOXACIN AND DEVELOPMENT OF COMPETITIVE ELISA ENR2KIT

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1 ,22 (6) : Journal of Nuclear Agricultural Sciences : (2008) ELISA 1, (11, ;21, ; 31, ;41, ) : (BSA2ENR), ELISA, ELISA (competitive ELISA ENR2Kit),: 2, IgG 1,4G12B3 ENR mab ELISA 1 : , ( Ka) LΠmol ; ELISA 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 ; 21 College of Bioengineering, Henan University of Technology, Zhengzhou ; 31 The Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, Henan ; 41 College of Animal Sciences, Henan Institute of Science and Technology, Xinxiang, Henan ) 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 in ascites. Isotype of the two mab was IgG 1. ENR mab had a high affinity constant ( Ka) with LΠmol. The ENR2Kit had the linear detection range of 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. : : :, (2006BAK02A21) : (19732),,,,, E2mail com. cn : (19602),,,,,E2mail com. cn ; (19482),,,,, E2mail com ' China Academic Journal Electronic Publishing House. All rights reserved.

2 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 g NaHCO g 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) ( ENR),,, [5,6 ] ( ENR2, BSA) , BalbΠc,50 g 012 mlπ,,,,,3 ( ENR mab), 3 1, ELISA ( competitive ELISA ENR (pab) ELISA ENR2Kit), ELISA ENR pab ENR ( IC 50 ), 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 d,,,, 25 g 4 5,,,,, (5 ),,, 37, 1min 1ml PEG1500, 90s 15ml (30s 1ml,30s 3ml,30s 11ml) 37 5min,, HAT,

3 () 96,HAT 10 HT, 14, ELISA ELISA, 24,7d,, 1 HT 5-7d,, 24,,, Πml,,14d rpm 30min,, ENR mab ELISA ( Ka) [7,9 ] Ig 1 ELISA ENR2Kit ( HRP2ENR) [5 ], ELISA, ELISA ENR mab HRP2ENR [8 ] ENR2Kit ENR mab HRP2ENR ELISA [10 ], PBS ( ), 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), 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 ENR ENR,ELISA IC 50, ENR mab ENR IC 50 IC 50 (CR %), [12 ] :1g 1ml ( PBS = 1 1),, 5s, 30min, rpm 1h, PBS 1 10, gπl ; [10 ],0101molΠL PBS %,, gπl 6, (CV) PBS 5,ENR2Kit, d ENR2Kit ELISA, ( IC 50 ) d,,1 10 ELISA, , 10 ELISA, IC gπl d 2,IC gπl 2 (4G1 5D2)

4 6 ELISA 901 4G Πml,5D Πml HT,4G1 1112, 5D d 70 ELISA, 70 OD ,ELISA, IC 50 IC gπl d,ic gπl(4g12g1 4G12B3), 4G12G1 4G12B3,, 50 %, ELISA,4G12B LΠmol,4G12G LΠmol, 1, [13,14 ], Ig IgG1 4G12B3 ELISA IC gπl 2 ENR24G12B3 ENR Fig. 2 Inhibitory curve of 4G12B3 mab , 0101 %, 1 4G12B3 Table 1 The cross reactivity of 4G12B3 mab inhibitors and multi antibiotics IC 50 ( gπl) cross2reactivity( %) enrofloxacin ciprofloxacin danofloxacin difloxacin norfloxacin sulfadiazine chloramphenicol streptomycin gentamycin ampicillin 1 Fig. 1 Association constant ( Ka) curve of ENR mab ENR mab HRP2ENR,ENR mab ; HRP2 ENR ENR mab ENR 2, Y = X , R 2 = ELISA 20, 1102, ;LOD (B0-2s)ΠB0, LOD = 0105 gπl, ENR2Kit 0105 gπl ENR2Kit gπl ENR mab ENR , 7718 % 8615 %,8115 %,CV 514 % 9181 %, 7162 % ; 83 % 9117 %, 8716 %,CV3155 % 814 %,611 % ; 8014 % 8918 %, 8413 %, CV 513 % %,7165 % ; 9015 % 9814 %,95 %,CV 416 % 915 %, 618 % ENR2Kit IC 50 :PBS 111 gπl 1145 gπl 210 gπl 2161 gπl 2194 gπl,, IC 50, ENR2Kit,

5 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% ELISA Fig. 3 Effect of different sample diluted solution on competitive ELISA ENR2Kit , ELISA, IC gπl, R ,, ,,, (BSA) (OVA), BSA,OVA ELISA, [5,6,15 ], EDC NHS,, ;,,,,, 1 29, , 4 ELISA ELISA, 1 10, , IC gπl, ( ),, ELISA ELISA,,,,, 312 ELISA PEG, HAT,7d, 90 %, 15 % ELISA ELISA,,,2 4G12B3 ENR mab ELISA , ( Ka) LΠmoL, ELISA gπl, ( IC 50 ) 111 gπl, ( CR %) 0101 %,,, ;,, ;,,, ENR, PBS 2, 86 % 99 % ;,PBS(VΠV = 1 1),PBS 1 10,

6 Journal of Nuclear Agricultural Sciences 2008,22 (6) : , 78 % 95 % 10 %,, ELISA, S. Bucknall,, ELISA, IC gπl [6 ] ; Hiroo Watanabe ELISA, 1 gπl,10ug/ kg, 8410 % 9910 % 7214 % 9210 % [5 ] ;, gπl, IC gπl [15 ], ELISA, IC gπl, ELISA gπl,,,,,,,, : [ 1 ] Hong J, Kim J M, Jung W K, Kim S H, Bae W, Koo H C, Gil J, Kim M, Ser J, Park Y H. Prevalence and antibiotic resistance of Campylobacter spp. isolated from chicken meat, pork, and beef in Korea, from 2001 to 2006.J Food Prot,2007,70 (4) : [ 2 ] Nelson J M, Chiller T M, Powers J H, Angulo F J. Fluoroquinolone2 resistant Campylobacter species and the withdrawal of fluoroquinolones from use in poultry : a public health success story. Clin Infect Dis, 2007,44 (7) : [ 3 ] Durden DA, MacPherson T. Quantitationand validationof fluoroquinolones in eggs using liquid chromatographyπtandem mass spectrometry. J AOAC Int,2007,90 (2) : [ 4 ] Zhao S, Jiang H, Li X, Mi T, Li C, Shen J. Simultaneous determination of trace levels of 10 quinolones in swine, chicken, and shrimp muscle tissues using HPLC with programmable fluorescence detection.j Agric Food Chem,2007,55 (10) : [ 5 ] Watanabe H, Satake A, Kido Y, Tsuji A. Monoclonal2based enzyme2 linked immunosorbent assays and immunochromatographic assay for enrofloxacin in biological matrices. Analyst,2002,127 : [ 6 ] Bucknall S, Silverlight J, Coldham N, Thorne L, Jackman R. Antibodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products. Food Additives and Contaminants,2003,20 (3) : [ 7 ],,.. :,1998 [ 8 ],.. :,2000 [ 9 ] Batty J D, Beatty B G, Wlanos W G. Measurement of monoclonal antibody affinity by non2competitive enzymeimmunoassay. Immunology Methods, 1987 (100) : Journal of [10 ],,,,,,. ELISA. (),2007,35 (4) :46 50 [11 ],,,,,,,. ELISA.,2003,39 (6) :6 8 [12 ] Huet A, Charlier C, Tittlemier, S Singh, G Benrejeb S, Delahaut P. Simultaneous determination of ( Fluoro) quinolone antibiotics in kidney, marine products, eggs, and muscle byenzyme2linked immunosorbent assay ( ELISA).J Agric Food Chem,2006,54 : [13 ] James W G. Monoclonal antibodies : Principles and Practice. New York : Academic Press, 1983, [14 ],,,,,.., 2006,20 (4) : [15 ] Jiuhua Duan, Zonghui Yuan. Development of an Indirect Competitive ELISA for CiprofloxacinResidues in Food Animal Edible Tissues. J Agric Food Chem,2001,49 : [16 ],,,. ELISA.,2006,40 (11) :

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