Genetically modified organisms (GMOs) is an organism genetic material has been altered to contain a segment of DNA from another organism. GM crops have become part of the global food industry, and South Korea has become one of the world's top two importers of GMO largely. In this study, monoclonal (...
Genetically modified organisms (GMOs) is an organism genetic material has been altered to contain a segment of DNA from another organism. GM crops have become part of the global food industry, and South Korea has become one of the world's top two importers of GMO largely. In this study, monoclonal (mAb) and polyclonal antibodies (pAb) specific to PAT and CP4-EPSPS proteins were produced from mouse ascites and rabbit antiserum, respectively, and developed sandwich ELISA and strip immunosensor for the rapid detection of PAT and CP4-EPSPS in maize and soybean.
1. Genomic DNA extracted from GM maize and soybean was amplified with a specific primer by PCR and amplified DNAs were cloned into pET-15b which is E. coli expression vector. The cloned vector was then transformed into competent E. coli BL21 (DE3) cell. Culture of the transformed E. coli BL21 (DE3) were induced proteins by the addition of IPTG and target proteins (PAT and CP4-EPSPS) as antigens was purified by Ni-NTA affinity chromatography.
2. A 7-8 weeks old BALB/c mice were immunized with recombinant PAT and CP4-EPSPS protein, and the titration of anti-sera from immunized mice was measured. After cell fusion and cloning, hybridomas producing specific mAbs to PAT (hybridoma No.: PAT-7 and PAT-12) and CP4-EPSPS (hybridoma No.: CP4-2 and CP4-3) were obtained and mAbs specific to PAT and CP4-EPSPS were produced. In addition, rabbits were respectively immunized with PAT and CP4-EPSPS proteins to produce pAbs that can be used as a capture antibody in strip immunosensor. The blood was obtained from heart of the rabbits by heart puncture method and pAbs specific to PAT and CP4-EPSPS were produced.
3. In this study, sandwich ELISAs and strip immunosensors were developed for the rapid detection of PAT and CP4-EPSPS proteins using generated mAbs and pAbs. First, sandwich ELISAs for PAT and CP4-EPSPS proteins were developed two mAb were selected out of 10 and 8 hybridoma cells and used as detector (PATmAb 7 and CP4-EPSPSmAb 2) and capture antibody (PATmAb 12 and CP4-EPSPSmAb 3) for PAT and CP4-EPSPS, respectively. PATmAb 7 and CP4-EPSPSmAb 2 were then conjugated with horseradish peroxides (HRP) and used as tracer in the sandwich ELISA. The detection limits of the established sandwich ELISA methods were 0.3 and 0.45 ng/ml for PAT and CP4-EPSPS, respectively. No cross-reaction to other GMO proteins was observed in the both sandwich ELISA methods.
4. Second, strip immunosensors based on gold nanoparticle (GNP)-antibody conjugates were developed for the rapid detection of PAT and CP4-EPSPS proteins. The same antibody pairs used for the sandwich ELISAs were used for the strip immunosensors. PATmAb 7 and CP4-EPSPSmAb 2 were conjugated with GNP and used as tracer for strip immunosensors. Strip immunosensors for PAT and CP4-EPSPS proteins were optimized and validated. The detection limits of the both immunosensors were all 0.5 ng/150 μl. No cross-reaction to other GMO proteins was observed. In addition, a strip immunosensor for simultaneous detection of PAT and CP4-EPSPS protein were developed using the same conditions for the individual strip immunosensor. The limits of strip immunosensor for simultaneous detection of PAT and CP4-EPSPS were all 1 ng/150 μl. Several stabilizing reagents such as bovine serum (BSA), sucrose, sodium azide, and the mixture of BSA and sucrose were tested for extending the self-life of the strip immunosensors. The strip immunosensors treated with the mixture of 5% BSA and sucrose were stable for 3 months at room temperature and 4℃.
5. Soybean, maize and processed foods contains either soybean or maize were analyzed by the strip immunosensor and sandwich ELISA methods. A total of 123 samples (soybean and its processed foods, 63; maize and its processed foods, 60) were tested by the strip immunosensors first, and the results were compared to those of commercial strip kits and the sandwich ELISA. Three methods provided the same results. The results showed that immunosensors developed in this study were highly specific and sensitive like commercial kits, and could be used for the rapid monitoring of GM maize and soybean. For the qualitative analysis, the strip immunosensor was better than instrumental analysis and PCR. On the other hand, the sandwich ELISAs are thought to be a suitable for the quantitative analysis method to detect GMOs.
Genetically modified organisms (GMOs) is an organism genetic material has been altered to contain a segment of DNA from another organism. GM crops have become part of the global food industry, and South Korea has become one of the world's top two importers of GMO largely. In this study, monoclonal (mAb) and polyclonal antibodies (pAb) specific to PAT and CP4-EPSPS proteins were produced from mouse ascites and rabbit antiserum, respectively, and developed sandwich ELISA and strip immunosensor for the rapid detection of PAT and CP4-EPSPS in maize and soybean.
1. Genomic DNA extracted from GM maize and soybean was amplified with a specific primer by PCR and amplified DNAs were cloned into pET-15b which is E. coli expression vector. The cloned vector was then transformed into competent E. coli BL21 (DE3) cell. Culture of the transformed E. coli BL21 (DE3) were induced proteins by the addition of IPTG and target proteins (PAT and CP4-EPSPS) as antigens was purified by Ni-NTA affinity chromatography.
2. A 7-8 weeks old BALB/c mice were immunized with recombinant PAT and CP4-EPSPS protein, and the titration of anti-sera from immunized mice was measured. After cell fusion and cloning, hybridomas producing specific mAbs to PAT (hybridoma No.: PAT-7 and PAT-12) and CP4-EPSPS (hybridoma No.: CP4-2 and CP4-3) were obtained and mAbs specific to PAT and CP4-EPSPS were produced. In addition, rabbits were respectively immunized with PAT and CP4-EPSPS proteins to produce pAbs that can be used as a capture antibody in strip immunosensor. The blood was obtained from heart of the rabbits by heart puncture method and pAbs specific to PAT and CP4-EPSPS were produced.
3. In this study, sandwich ELISAs and strip immunosensors were developed for the rapid detection of PAT and CP4-EPSPS proteins using generated mAbs and pAbs. First, sandwich ELISAs for PAT and CP4-EPSPS proteins were developed two mAb were selected out of 10 and 8 hybridoma cells and used as detector (PATmAb 7 and CP4-EPSPSmAb 2) and capture antibody (PATmAb 12 and CP4-EPSPSmAb 3) for PAT and CP4-EPSPS, respectively. PATmAb 7 and CP4-EPSPSmAb 2 were then conjugated with horseradish peroxides (HRP) and used as tracer in the sandwich ELISA. The detection limits of the established sandwich ELISA methods were 0.3 and 0.45 ng/ml for PAT and CP4-EPSPS, respectively. No cross-reaction to other GMO proteins was observed in the both sandwich ELISA methods.
4. Second, strip immunosensors based on gold nanoparticle (GNP)-antibody conjugates were developed for the rapid detection of PAT and CP4-EPSPS proteins. The same antibody pairs used for the sandwich ELISAs were used for the strip immunosensors. PATmAb 7 and CP4-EPSPSmAb 2 were conjugated with GNP and used as tracer for strip immunosensors. Strip immunosensors for PAT and CP4-EPSPS proteins were optimized and validated. The detection limits of the both immunosensors were all 0.5 ng/150 μl. No cross-reaction to other GMO proteins was observed. In addition, a strip immunosensor for simultaneous detection of PAT and CP4-EPSPS protein were developed using the same conditions for the individual strip immunosensor. The limits of strip immunosensor for simultaneous detection of PAT and CP4-EPSPS were all 1 ng/150 μl. Several stabilizing reagents such as bovine serum (BSA), sucrose, sodium azide, and the mixture of BSA and sucrose were tested for extending the self-life of the strip immunosensors. The strip immunosensors treated with the mixture of 5% BSA and sucrose were stable for 3 months at room temperature and 4℃.
5. Soybean, maize and processed foods contains either soybean or maize were analyzed by the strip immunosensor and sandwich ELISA methods. A total of 123 samples (soybean and its processed foods, 63; maize and its processed foods, 60) were tested by the strip immunosensors first, and the results were compared to those of commercial strip kits and the sandwich ELISA. Three methods provided the same results. The results showed that immunosensors developed in this study were highly specific and sensitive like commercial kits, and could be used for the rapid monitoring of GM maize and soybean. For the qualitative analysis, the strip immunosensor was better than instrumental analysis and PCR. On the other hand, the sandwich ELISAs are thought to be a suitable for the quantitative analysis method to detect GMOs.
AI-Helper
※ AI-Helper는 부적절한 답변을 할 수 있습니다.