ELISA (, 100021) :,, ELISA,AFB 1 - BSA, 01062 5gΠml,1 :116 10 6 B + G0113 ngπml, 1150gΠkg 012620100 ngπml, y = - 01446 3 x + 01353 2 ( R 2 = 01991 5) ; B + G 50 % 2108 ngπml ;2 94156 %112105 %,2 87150 %85160 % B 1 B 2 G 1 G 2 100 % 5715 % 104 %19 %, : ;,;, [3 ] [ 8,9 ] 9, : 13,13π ; 11,11π ; 9,9π 7 9,,C 30 C 18, C 30, C 30,,,, [1 ] Young A J, Britton G, eds. Carotenoids in photosynthesis [M]. London : Chapman and Hall, 1993. 4092416. [2 ] Britton G, Liaaen2Jensen S, Pfander H, eds. Carotenoids (1A) Basel [ M ]. Boston and Berlin : Birkhguser, 1995. 1692284. [3 ],,,. [M]. :,2005. 1172129. [4 ],,. [J ].,2002, (5) : 722 82. [5 ] Riemersma R A, Wood D A, Macintyre C C, et al. Risk of angina pectoris and plasma concentrations of vitamins A, C, E and carotene[j ]. Lancer, 1991, (337) : 125. [6 ] Tsukida K, Saiki K, Takii, et al. Separation and determina2 tion of cisπtrans2 2carotenes by high2performance liquid chro2 matography[j ]. J Chromatogr,1982, (245) : 3592364. [7 ] Lin C H, Chen B H. Determination of carotenoids in tomato juice by liquid chromatography [ J ]. J Chromatogr, 2003, (1012) :1032109. [8 ] Emenhiser C, Sander L C. Separation of geometrical carote2 noid isomers in biological extracts using a polymeric C 30 col2 umn in reversed2phase liquid chromatography [ J ]. J Agric Food Chem,1996, (44) :388723893. [9 ] Emenhiser C, Sander L C, Schwartz S J. Capability of a polymeric C 30 stationary phase to resolve cis2trans carotenoid isomers in reversed phase liquid chromatography[j ]. J Chro2 matogr A, 1995, (707) : 2052216. [ :2006-03 - 22 ] :R15 ;O657172 :A :1004-8456 (2006) 04-0289 - 04 :(2001BA804A20) : : 292 2006 18 4
Quantitative Analysis of Aflatoxins by EL ISA J IANG Tao, LIU Zhen, ZHENGJia, LI Min, J I Rong (National Institute for Nutrition and Food Safety, Chinese CDC, Beijing 100021, China) Abstract : To detect the level of contamination of total aflatoxins in grains, a rapid, specific and quantitative ELISA2based method was developed using monoclonal antibodies against Aflatoxins. The coating antigen was AFB 1 2BSA and the concentration was 01062 5gΠml. The working titre of antibody was 1 : 116 10 6 1 For the standard Aflatoxin (B + G), the limit of detection was 0113 ngπml ; for samples, the limit of detection was 1150gΠkg ; the linear range was 012620100 ngπml and the linear equation was y = - 01446 3 x + 01353 2 ( R 2 = 01991 5). The 50 % inhibition concentration for Aflatoxin (B + G) was 2108 ngπml. The recovery rates of spiked maize on the level of 2110gΠkg and 10140gΠkg were 94156 % and 112105 % respectively. For peanut recovery rates on the level of 2100gΠkg and 4100gΠkg were 87150 % and 85160 %, respectively. The cross reaction rates with aflatoxin B 1, B 2, G 1, G 2 were 100 %, 5715 %, 104 %, 19 % respectively, and there were no cross reactions with other my2 cotoxins. Key word : Aflatoxins ;Antibody Monoclonal ; Enzyme2Linked Immunosorbent Assay B 1 + B 2 + G 1 + G 2,, 5gΠkg [1 ],, ;;,,, ( ELISA),, ELSIA ELISA 2002, ELISA, B, 1 111 CO 2 ( Queue ) () ( ) (SUNRISE) ( Mettler ) ( Hermle ) (BIO - RAD) AFB 1 - BSA (B + G) (Aflatoxin B + G) B 1 (Aflatoxin B 1 ) B 2 (Aflatoxin B 2 ) G 1 (Aflatoxin G 1 ) G 2 (Aflatoxin G 2 ) T - 2 ( T - 2 toxin) A(Ochratoxin A) ( Patulin) (Deoxynivalenol,DON) (Zearalenone,ZEN) ( Keyhole limpet he2 mocyanin,klh) (Bovine serum albu2 min, BSA) B 1 ( IgM IgG IgA IgD IgG 1 IgG 2a IgG 2b IgG 3 ), Sigma ; RPMI1640 HAT HEPES ( ) (DMF) 20 ( Tween 20) ( PEG, MW 5000),Gibco ; IgG, 30 % H 2 O 2 ELISA 0105 molπl, ph 916 ; PBS - T, 0105 % Tween20 0105 molπl PBS ( ) ; 011 molπl 012 molπl,ph 510 ; 10 mg TMB 1 ml, 75 l,10 ml 10 l H 2 O 2 ; 2 molπl H 2 SO 4 011 %( ) BSA 0105 molπl PBS Sp2Π0, 8 - BalBΠc 112 11211 BalBΠc,, ELISA 293
[2 ] 11212 ; SDS - PAGE [3 ] ; IgG BCA ; ELISA [2 ] :AFB 1 - BSA, 1 10 000,Sp2Π0,(A - ) / ( A - ) 211 ; ELISA [4 ], B 1 B 2 G 1 G 2 (DON) A(OA) B 1 T - 2 BSA 2 113 ELISA [5 ] 211 11311 (), 1 :2156 10 7, ELISA,1 1 116 10 6 116 10 6 ELISA, B 1 B 2 G 1 G 2 50 % AFB 1 - BSA, 1120 2109 1115 6131 ngπml ( 2 3 4 5) 01062 5gΠml, 120 l,(b + G) 0 01026 01052 0113 0126 0152 1130 2160 5120 13100 26100 52100 130100 260100 ngπml, (1 116 10 6 ), 3, 3, 1, B + G 0113 ngπml 30, ), 30 min 10 (gπkg) = CDXΠW : C (ngπml), ; D (ml) ; X ; W (g) 11312 () () 11313 ELISA HPLC ELISA AOAC HPLC, IgG 1, 1152 10-10 molπl 150 kd, IgG10 gπl B 1 B 2 G 1 G 2 100 % 5715 % 104 %19 %, T - 2 A B 1 011 % 1 (B + G) ( ELISA) 11312, 2, 2110 10140gΠkg, 2100 4100gΠkg 5 90 % 250500m 5 g 100 ml, 015 g NaCl 25 ml (70 + 2 B 1 ( ELISA) 3 B 2 ( ELISA) 294 2006 18 4
4 G 1 ( ELISA) 21213 BSA 0 0110 20 0150 1100 2100 5100 101002010050100100100200100500100 1 000100 2 000100 ngπml,,50 %,,BSA 1 %, 21214 ELISA HPLC ELISA AOAC HPLC, 3t, P > 0105, 3 ELISA HPLC gπg 1 2 3 4 5 6 ELISA 6127 6155 6141 16114 16148 16118 5 G 2 ( ELISA) 212 ELISA 21211, 1 : 116 10 6,AFB 1 - BSA 01062 5 gπml 21212 (B + G) 2110 10140gΠkg 2, 1 (B + G) 2100 4100gΠkg 2, 2 1 (B + G) % 1 2 1 96177 117124 2 98113 103186 3 94156 116136 4 86158 113138 5 96177 109140 94156 112105 RSD 4191 4192 :1 :2110 gπkg ; 2 : 10140 gπkg 2 (B + G) % 1 2 1 80137 97196 2 75167 96161 3 90103 76192 4 103186 75176 5 87158 80177 87150 85160 RSD 10178 10183 :1 :2100 gπkg ;2 : 4100 gπkg HPLC 8160 7120 7112 14171 15120 15120 21215 ELISA Biopharm 4 4 ELISA Biopharm ELISA ELISA Kit of Biopharm gπkg 1150 1175 % 9419 85 % 719 15 % AFB 1 10010 100 AFB 2 5715 200 AFG 1 104 15 AFG 2 19 16 4, ELISA, Biopharm,, ELISA, ;Biopharm,, B 1 B 2 G 1 G 2, ELISA 0126gΠkg, 1150gΠkg, 2 94156 %112105 %, 2 87150 %85160 % ELISA 295
Cry1Ie 1 1 1 2 2 1 (11, 100021 ; 21, 100094) : Cry1Ie, Cry1Ie 72 ICR Cry1Ie (833gΠkg BW d - 1 30 d), ( ) ( ) Cry1Ie,, Cry1Ie : ; ; ; Effects of Cry1Ie Protein Expressed in E coli. on Biochemical Indicators and Flora of Intestinal Tract in Mice ZHANG Xiao2xia, YANG Bao2lan, YAN Wei2xing, LIU Yun2jun, WANG Gou2ying, XU Hai2bin (National Institute for Nutrition and Food Safety, Chinese CDC, Beijing 100021, China) Abstract : Cry1Ie expressed protein is used as a model protein to examine its effects on biochemical indicators and flora of intesti2 nal tract in mice. 72 ICR mice were randomly divided into one experimental group and two control groups. The experimental group was treated with 833gΠkg BW d - 1 Cry1Ie expressed protein. The two control groups were treated with solution of bovine serum albumin and culture solution of E coli. with control plasmid without Cry1Ie, respectively. After 30 days, body weight, biochemi2 cal indicators (ASTALTTPALBBUNCRETRIGCHOL GLU) and numbers of flora of intestinal tract (enteric ba2 cilli, enterococci, Bacillus bifidus, lactobacilli) were examined. There were no significant differences between experimental group and control groups in body weight, biochemical indicators and numbers of flora of intestinal tract. In the present study, the Cry1Ie expressed protein had no toxicity to experimental animals. These results offer basic information for safety evaluation of new transgenic Cry1Ie expressed food, and establish foundation for food safety assessment of exogenous proteins derived from modern biological technique. Key word : Proteins ; Escherichia coil ; Blood Chemical Analysis ; mice, (B + G), ELISA, [1 ] GBΠT 5009. 23 2003. B 1 B 2 G 1 [ S]. G 2 [2 ],. [M]. 2. :,2002. 2802282. [3 ],,.J,E F, T,. [M]. 2. :,1996. 8802887. [4 ],,,. A [J ]., 2004,16 (1) 14217. [5 ],,,. A [J ].,2004,20 (5) : 5562559. [ :2006-03 - 22 ] :R15 ;R379 ;Q949132 :A :1004-8456 (2006) 04-0292 - 05 : (J002C2003) : : 296 2006 18 4