This study reported that a hapten of imidacloprid, 1-[（6-Carboxylethylthio-3- pyridinyl）methyl]-N -nitro-imidazolidinimine was synthesized using technical material of imidacloprid to react with 3-mercaptopropionic a...This study reported that a hapten of imidacloprid, 1-[（6-Carboxylethylthio-3- pyridinyl）methyl]-N -nitro-imidazolidinimine was synthesized using technical material of imidacloprid to react with 3-mercaptopropionic acid in hot alkaline solution. The hapten was conjugated to bovine serum albumin （BSA） and ovalbumin （OVA） to form the immuno-antigens and the coating antigen, respectively. The antibody with high titer （2.56 × 10^4） was obtained after immuning rabbits. The results showed that the antibody had a high affinity to imidacloprid tested by enzyme-linked immunosorbent assay （ELISA）, which IC50 and IC20 were 20.7 and 1.1 μg L^-1; and the cross reactibilities to those compounds related with imidacloprid were less than 1.4%.展开更多
A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Tw...A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.展开更多
Mixed anhydride(MA)was used to conjugate ractopamine(RAC)to BSA and obtained artificial antigen BSA-RAC identified by UV and SDS-PAGE.Balb/c mice were immunized with BSA-RAC and hybridoma lines that secrete RAC monocl...Mixed anhydride(MA)was used to conjugate ractopamine(RAC)to BSA and obtained artificial antigen BSA-RAC identified by UV and SDS-PAGE.Balb/c mice were immunized with BSA-RAC and hybridoma lines that secrete RAC monoclonal antibody(mAb)were generated with cell fusion.A ciELISA kit for detection of RAC(RAC-Kit)was developed with RAC mAb and its performance were tested.The results indicated that BSA-RAC was successfully synthesized and its conjugation ratio of RAC to BSA was about 24.5∶1.Three hybridoma lines were filtered and the best one was 4D8-3E11,its affinity constant(Ka)was 1.65×1010 L/mol.The limit of detection of RAC-Kit was 0.5 ng/ml and its detection range was 0.5-184 ng/ml.The mean recoveries of RAC spiked in feed were 85.6% and in swine urine were 88.6%.The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%.It had 9.4% cross-reactivity(CR%)to dobutamine and little or no CR to other compounds.The validity of RAC-Kit in 4 ℃ was in 180 d.展开更多
基金the National Natural Science Foundation of China （30370944）.
文摘This study reported that a hapten of imidacloprid, 1-[（6-Carboxylethylthio-3- pyridinyl）methyl]-N -nitro-imidazolidinimine was synthesized using technical material of imidacloprid to react with 3-mercaptopropionic acid in hot alkaline solution. The hapten was conjugated to bovine serum albumin （BSA） and ovalbumin （OVA） to form the immuno-antigens and the coating antigen, respectively. The antibody with high titer （2.56 × 10^4） was obtained after immuning rabbits. The results showed that the antibody had a high affinity to imidacloprid tested by enzyme-linked immunosorbent assay （ELISA）, which IC50 and IC20 were 20.7 and 1.1 μg L^-1; and the cross reactibilities to those compounds related with imidacloprid were less than 1.4%.
基金This work was financially supported by National Natural Science Foundation of China （20447003）.
文摘A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.
基金Supported by the Key Project of National Science and Technology Surporting Plan during 11th-Five-Year of China(2006BAK02A21/1)~~
文摘Mixed anhydride(MA)was used to conjugate ractopamine(RAC)to BSA and obtained artificial antigen BSA-RAC identified by UV and SDS-PAGE.Balb/c mice were immunized with BSA-RAC and hybridoma lines that secrete RAC monoclonal antibody(mAb)were generated with cell fusion.A ciELISA kit for detection of RAC(RAC-Kit)was developed with RAC mAb and its performance were tested.The results indicated that BSA-RAC was successfully synthesized and its conjugation ratio of RAC to BSA was about 24.5∶1.Three hybridoma lines were filtered and the best one was 4D8-3E11,its affinity constant(Ka)was 1.65×1010 L/mol.The limit of detection of RAC-Kit was 0.5 ng/ml and its detection range was 0.5-184 ng/ml.The mean recoveries of RAC spiked in feed were 85.6% and in swine urine were 88.6%.The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%.It had 9.4% cross-reactivity(CR%)to dobutamine and little or no CR to other compounds.The validity of RAC-Kit in 4 ℃ was in 180 d.
