In this stHdy, lateral-flow chromatography with fluorescence was applied for the development of proteins and DNA assays. The protein and DNA quantitative assay strip is composed of a sample pad, an absorption pad for the generation of capillary action, and a nitrocellulose membrane where immune reac...
In this stHdy, lateral-flow chromatography with fluorescence was applied for the development of proteins and DNA assays. The protein and DNA quantitative assay strip is composed of a sample pad, an absorption pad for the generation of capillary action, and a nitrocellulose membrane where immune reaction of antigen/antibody and hybridization of DNA occur. For the protein quantitative assay system, AFP, CEA, PSA, TSH, and HbAlc strips were fsbricated. For the measurement of each item specific monoclonal antibody and polyclonal antibody were used of the detection system by sandwich and competition format. This system is composed of a test strip with dispensed antibody, a disposable cartridge, detector buffer, and a laser-fluorescence scanner for the measurement fluorescence intensity. The calibration curves displayed a linearity within the range of AFP : 1-350ng/ml, CEA:4-200ng/ml, PSA: 1-50ng/ml, TSH : 1-40mIU/L and HbAlc : 0.25-2mg/ml, and coefficient correlations of the curves were AFP R=0.997, CEA R=0.995, PSA R=0.997, TSH R= 0.998, and HbAlc R= 0.996. Intra-and inter-assay imprecisions were determined to be within 10% of CVs. Analytical recovery was above 95% at 3 different concentrations and the detection limit was of AFP : 0.48ng/mL, CEA : 0.5ng/ml, PSA : 0.184ng/ml, and TSH : 0.868mIU/L. The assay system correlated well with full automatic instruments for quantification of AFP(R=0.989, slope=0.908), CEA(R=0.990, slope=0.973), PSA(R=0.993, slope=0.982), TSH(R=0.97, slope=0.94), HbAlc(R=0.963, slope=1.0). DNA analysis system was developed for the detection of HIV, HCV, and HPV DNA. The strip contained immobilized DNA as a capture line and target DNA was labeled with cy5/biotin for signaling. The samples containing a target DNA were applied onto the sample pad and scanned with a GSI fluorescence scanner and/or a fluorescence reader(i-chroma). Though the hybridization reaction occurs in a short time, the target DNA could be captured on the test line. Furthermore, there appears to be little cross hybridization between the different sequences(HIV/HCV, HPV 16/18). To confirm that the scanning results actually reflect the abundance of the amplified DNA by PCR, we checked the DNA band on an agarose gel. As expected, the intensity of the peak showed a good correlation with the DNA band. In addition, when the biotin-labeled primer was used instead of Cy5-labeled primer, the analytical sensitivity was much improved. Even though we could not see any DNA band on a gel, the scanner displayed discrete peak on the strip. On the basis of these result, it was concluded that DNA analysis system developed in this study is better in analytical sensitivity than in the electrophoresis method. In this study, protein and DNA quantification systems are a convenient and fast system and can be used a good tool for detection and quantification of analyte for diagnostic of disease in the specimen. Futhermore, the portable system allows us to perform the tests on a sampling site in any places without bringing the samples to a laboratory.
In this stHdy, lateral-flow chromatography with fluorescence was applied for the development of proteins and DNA assays. The protein and DNA quantitative assay strip is composed of a sample pad, an absorption pad for the generation of capillary action, and a nitrocellulose membrane where immune reaction of antigen/antibody and hybridization of DNA occur. For the protein quantitative assay system, AFP, CEA, PSA, TSH, and HbAlc strips were fsbricated. For the measurement of each item specific monoclonal antibody and polyclonal antibody were used of the detection system by sandwich and competition format. This system is composed of a test strip with dispensed antibody, a disposable cartridge, detector buffer, and a laser-fluorescence scanner for the measurement fluorescence intensity. The calibration curves displayed a linearity within the range of AFP : 1-350ng/ml, CEA:4-200ng/ml, PSA: 1-50ng/ml, TSH : 1-40mIU/L and HbAlc : 0.25-2mg/ml, and coefficient correlations of the curves were AFP R=0.997, CEA R=0.995, PSA R=0.997, TSH R= 0.998, and HbAlc R= 0.996. Intra-and inter-assay imprecisions were determined to be within 10% of CVs. Analytical recovery was above 95% at 3 different concentrations and the detection limit was of AFP : 0.48ng/mL, CEA : 0.5ng/ml, PSA : 0.184ng/ml, and TSH : 0.868mIU/L. The assay system correlated well with full automatic instruments for quantification of AFP(R=0.989, slope=0.908), CEA(R=0.990, slope=0.973), PSA(R=0.993, slope=0.982), TSH(R=0.97, slope=0.94), HbAlc(R=0.963, slope=1.0). DNA analysis system was developed for the detection of HIV, HCV, and HPV DNA. The strip contained immobilized DNA as a capture line and target DNA was labeled with cy5/biotin for signaling. The samples containing a target DNA were applied onto the sample pad and scanned with a GSI fluorescence scanner and/or a fluorescence reader(i-chroma). Though the hybridization reaction occurs in a short time, the target DNA could be captured on the test line. Furthermore, there appears to be little cross hybridization between the different sequences(HIV/HCV, HPV 16/18). To confirm that the scanning results actually reflect the abundance of the amplified DNA by PCR, we checked the DNA band on an agarose gel. As expected, the intensity of the peak showed a good correlation with the DNA band. In addition, when the biotin-labeled primer was used instead of Cy5-labeled primer, the analytical sensitivity was much improved. Even though we could not see any DNA band on a gel, the scanner displayed discrete peak on the strip. On the basis of these result, it was concluded that DNA analysis system developed in this study is better in analytical sensitivity than in the electrophoresis method. In this study, protein and DNA quantification systems are a convenient and fast system and can be used a good tool for detection and quantification of analyte for diagnostic of disease in the specimen. Futhermore, the portable system allows us to perform the tests on a sampling site in any places without bringing the samples to a laboratory.
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#유전 공학(생물학)[遺傳工學]
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