단일클론 항체를 사용하여 간편하고 신속하게 물질을 측정할 수 있는 금입자 면역크로마토그래피 법을 개발하고 이 방법을 hCG와 HBsAg을 검출하는데 응용하였다. 또한 결과를 정량화 하기 위한 시도로서 컴퓨터 상 분석을 실시하였으며 결과를 요약하면 다음과 같다. 1. 항 hCG항체를 분비하는 5개의 융합세포주를 확립하였으며 이중 2개는 β-hCG와 결합하는 항체를 분비하였으며 나머지 3개는 α-hCG와 결합하는 항체를 분비하였다. 2. 1% HAuCl_(4) 용액에 1% 구염산 나트륨을 7 ml, 3 ml, 1 ml, 0.5 ml을 각각 첨가 했을 때 20 nm, 40 nm, 80nm 및 150nm 크기의 금입자가 생성되었다. 3. 금입자 항체 복합체 제조시에 최적조건은 CKD-35 항체의 경우 pH 8, 항체농도 4㎍/ml 이였고 KS-CKD1 항체의 경우는 pH 6, 항체농도 4 ㎍/ml 이였다. (금입자 크기는 40 nm) 4. 면역크로마토그래피 스트립의 안정성을 시험 했을 때 상온, 50℃와 60℃에서는 2달, 그리고 ...
단일클론 항체를 사용하여 간편하고 신속하게 물질을 측정할 수 있는 금입자 면역크로마토그래피 법을 개발하고 이 방법을 hCG와 HBsAg을 검출하는데 응용하였다. 또한 결과를 정량화 하기 위한 시도로서 컴퓨터 상 분석을 실시하였으며 결과를 요약하면 다음과 같다. 1. 항 hCG항체를 분비하는 5개의 융합세포주를 확립하였으며 이중 2개는 β-hCG와 결합하는 항체를 분비하였으며 나머지 3개는 α-hCG와 결합하는 항체를 분비하였다. 2. 1% HAuCl_(4) 용액에 1% 구염산 나트륨을 7 ml, 3 ml, 1 ml, 0.5 ml을 각각 첨가 했을 때 20 nm, 40 nm, 80nm 및 150nm 크기의 금입자가 생성되었다. 3. 금입자 항체 복합체 제조시에 최적조건은 CKD-35 항체의 경우 pH 8, 항체농도 4㎍/ml 이였고 KS-CKD1 항체의 경우는 pH 6, 항체농도 4 ㎍/ml 이였다. (금입자 크기는 40 nm) 4. 면역크로마토그래피 스트립의 안정성을 시험 했을 때 상온, 50℃와 60℃에서는 2달, 그리고 상대습도 75%로 조절된 40℃ 에서는 1주일간 안정하였다. 5. hCG측정을 위한 면역크로마토그래피법의 검출한계는 12.5 mIU/ml이였고 구조가 유사한 hTSH, hLH, hFSH와 교채반응을 나타내지 않았다. 6. HBsAg 측정에 응용한 면역크로마토그래피법의 검출한계는 4ng/ml 이였고 임상실험결과 민감도는 97%, 특이도는 100%이였다. 7. 결과를 정량화 하기위한 컴퓨터 상 분석에서 검출가능범위는 10 mIU/ml에서 1000 mIU/ml 이였고 편차는 2.5%이였다. 또한 intra-assay와 inter-assay의 변이상수는 25 mIU/ml에서 2.03%, 2.84% 이였고, 100 mIU/ml에서 3.14%, 4.77%이며, 높은 농도인 350 mIU/ml에서 3.55%, 6.28% 이였다. 이상의 결과는 금입자와 단일글론항체를 이용하여 개발한 면역크로마토그래픽법이 임상적으로 사용될 수 있음을 나타내었다. 본 방법의 가장 큰 장점은 상온에서 보관이 가능하며 5분 이내에 신속하게 단 한번의 조작으로 고가의 장비없이 검사할수 있어 여러 분야에 응용할 수 있을 것으로 기대된다.
단일클론 항체를 사용하여 간편하고 신속하게 물질을 측정할 수 있는 금입자 면역크로마토그래피 법을 개발하고 이 방법을 hCG와 HBsAg을 검출하는데 응용하였다. 또한 결과를 정량화 하기 위한 시도로서 컴퓨터 상 분석을 실시하였으며 결과를 요약하면 다음과 같다. 1. 항 hCG항체를 분비하는 5개의 융합세포주를 확립하였으며 이중 2개는 β-hCG와 결합하는 항체를 분비하였으며 나머지 3개는 α-hCG와 결합하는 항체를 분비하였다. 2. 1% HAuCl_(4) 용액에 1% 구염산 나트륨을 7 ml, 3 ml, 1 ml, 0.5 ml을 각각 첨가 했을 때 20 nm, 40 nm, 80nm 및 150nm 크기의 금입자가 생성되었다. 3. 금입자 항체 복합체 제조시에 최적조건은 CKD-35 항체의 경우 pH 8, 항체농도 4㎍/ml 이였고 KS-CKD1 항체의 경우는 pH 6, 항체농도 4 ㎍/ml 이였다. (금입자 크기는 40 nm) 4. 면역크로마토그래피 스트립의 안정성을 시험 했을 때 상온, 50℃와 60℃에서는 2달, 그리고 상대습도 75%로 조절된 40℃ 에서는 1주일간 안정하였다. 5. hCG측정을 위한 면역크로마토그래피법의 검출한계는 12.5 mIU/ml이였고 구조가 유사한 hTSH, hLH, hFSH와 교채반응을 나타내지 않았다. 6. HBsAg 측정에 응용한 면역크로마토그래피법의 검출한계는 4ng/ml 이였고 임상실험결과 민감도는 97%, 특이도는 100%이였다. 7. 결과를 정량화 하기위한 컴퓨터 상 분석에서 검출가능범위는 10 mIU/ml에서 1000 mIU/ml 이였고 편차는 2.5%이였다. 또한 intra-assay와 inter-assay의 변이상수는 25 mIU/ml에서 2.03%, 2.84% 이였고, 100 mIU/ml에서 3.14%, 4.77%이며, 높은 농도인 350 mIU/ml에서 3.55%, 6.28% 이였다. 이상의 결과는 금입자와 단일글론항체를 이용하여 개발한 면역크로마토그래픽법이 임상적으로 사용될 수 있음을 나타내었다. 본 방법의 가장 큰 장점은 상온에서 보관이 가능하며 5분 이내에 신속하게 단 한번의 조작으로 고가의 장비없이 검사할수 있어 여러 분야에 응용할 수 있을 것으로 기대된다.
A simple and rapid Immunochromatographic Assay(ICA) system was developed using monoclonal antibodies and gold particle as a label this system was applied for the detections of hCG(human Chorionic Gonadotropin) and HBsAg(hepatitis B suface antigen). In addition, it computer image-and data-processing ...
A simple and rapid Immunochromatographic Assay(ICA) system was developed using monoclonal antibodies and gold particle as a label this system was applied for the detections of hCG(human Chorionic Gonadotropin) and HBsAg(hepatitis B suface antigen). In addition, it computer image-and data-processing procedure has been developed for the quantification of the results appeared in a ICA strip. The results obtained were summarized as follow; 1. It was established 5 hybridoma cell lines that produce monoclonal antibodies to hCG. 2 clones of them were specific to β-hCG subuints, while remaining other 3 clones were specific to α-hCG subuints. 2. When 7 ml, 3 ml, and 0.5 ml of 1% sodium citrate solution were added to 200 ml of 1% HAuC1_(4) solution, the size of gold particles, which was produced, was 20 nm, 40 nm, and 80 nm, 150 nm, respectively. 3. The optimal pH and concentration of antibody for the production of CKD-35 antibody-gold conjugate were pH 8 and 4 ㎍/ml when gold particle with a size of 40 nm was used. 4. Immunochromatographic assay strip was stable for 2 months at room temperature, 50℃ and 60℃. while it was stable for 1 week at 40℃ with 75%(relative humidity) 5. The detection limit of ICA for the measurement of hCG was 12.5 mIU/ml and it did not show cros-reactivities with hLH, hFSH, and hTSH. 6. ICA for the assay of HBs Ag in whole blood was evaluated in terms of detection limit, comparison studies with other known method and time required for the assay. 29 of clinicl positive samples (97%) were correlated each other between ICA and compared conventional HA and ELISA while 45 samples with negative results assayed by HA and ELISA showed also negative ones(100%). ICA procedure could be completed within 5 minutes wheras HA and ELISA requried more than one hour. The detection limits of ICA, HA and ELISA were found to be 4, 2 and 0.25 ng/ml, respectively 7. An image-and data processing procedure for the quantification of the strip assay was developed. The integrated optical density(IOD) of a gold line was measured after completion of the assay. The IOD was increased linearly with increasing concentration of hCG to be measured and the standard curve could be constructed from 10 mIU/ml to 1000 mIU/ml. The intra-assay and inter-assay precision was calculated from repeated analyses of the same sample on a number of different occasions. This study it was demonstrated that the colloidal gold label and specific monoclonal antibodies has been used successfuly for the assays of hCG and HBsAg. The great advantages of this metod are that the time required for the assay is very short ; nitrocellulose absorbed antibodies and gold particles is very stable even at room temperature and it does not require very expensive equipments. The principle presented in this study could be applied on the other systems such as HIV, HCV etc.
A simple and rapid Immunochromatographic Assay(ICA) system was developed using monoclonal antibodies and gold particle as a label this system was applied for the detections of hCG(human Chorionic Gonadotropin) and HBsAg(hepatitis B suface antigen). In addition, it computer image-and data-processing procedure has been developed for the quantification of the results appeared in a ICA strip. The results obtained were summarized as follow; 1. It was established 5 hybridoma cell lines that produce monoclonal antibodies to hCG. 2 clones of them were specific to β-hCG subuints, while remaining other 3 clones were specific to α-hCG subuints. 2. When 7 ml, 3 ml, and 0.5 ml of 1% sodium citrate solution were added to 200 ml of 1% HAuC1_(4) solution, the size of gold particles, which was produced, was 20 nm, 40 nm, and 80 nm, 150 nm, respectively. 3. The optimal pH and concentration of antibody for the production of CKD-35 antibody-gold conjugate were pH 8 and 4 ㎍/ml when gold particle with a size of 40 nm was used. 4. Immunochromatographic assay strip was stable for 2 months at room temperature, 50℃ and 60℃. while it was stable for 1 week at 40℃ with 75%(relative humidity) 5. The detection limit of ICA for the measurement of hCG was 12.5 mIU/ml and it did not show cros-reactivities with hLH, hFSH, and hTSH. 6. ICA for the assay of HBs Ag in whole blood was evaluated in terms of detection limit, comparison studies with other known method and time required for the assay. 29 of clinicl positive samples (97%) were correlated each other between ICA and compared conventional HA and ELISA while 45 samples with negative results assayed by HA and ELISA showed also negative ones(100%). ICA procedure could be completed within 5 minutes wheras HA and ELISA requried more than one hour. The detection limits of ICA, HA and ELISA were found to be 4, 2 and 0.25 ng/ml, respectively 7. An image-and data processing procedure for the quantification of the strip assay was developed. The integrated optical density(IOD) of a gold line was measured after completion of the assay. The IOD was increased linearly with increasing concentration of hCG to be measured and the standard curve could be constructed from 10 mIU/ml to 1000 mIU/ml. The intra-assay and inter-assay precision was calculated from repeated analyses of the same sample on a number of different occasions. This study it was demonstrated that the colloidal gold label and specific monoclonal antibodies has been used successfuly for the assays of hCG and HBsAg. The great advantages of this metod are that the time required for the assay is very short ; nitrocellulose absorbed antibodies and gold particles is very stable even at room temperature and it does not require very expensive equipments. The principle presented in this study could be applied on the other systems such as HIV, HCV etc.
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