Diagnostic and Biochemical Characterization of Hepatitis C Virus Envelope Protein Using Specific Monoclonal Antibodies
Iman Ali Mohamed El Demerdash Touni;
Abstract
Diagnostic and Biochemical Characterization of Hepatitis C Virus Envelope Protein Using Specific Monoclonal Antibodies
In this study, revival and propagation of cell lines of each of the four hybrids (7G9),(6D11),(3F6),(6E4) producing the monoclonal antibodies targetingHCV E1/E2 envelope proteins was accomplished.
All four supernatants of these hybrids showed positive results for the presence of the monoclonal antibodies targeting HCV E1/E2 envelope proteins by ELISA. The hybrid (7G9 ) showing the highest OD reading (2.6) while the positive control was (1.7) and the negative control was (0.4).
The supernatants were purified by the ammonium sulphate precipitation method to obtain the monoclonal antibodies.
The monoclonal antibodies (7G9),(6D11),(3F6),(6E4) were characterized by determining their isotypeswhich were found to be IgM/Kappa chain for all.
The protein content of each of them was also determined where (7G9) was 9.5mg/ml, (6D11) was 9.75mg/ml, (3F6) was 10.5mg/ml and (6E4) was 8.0 mg/ml.
The reactivity of the four monoclonal antibodies against the peptide antigens P1 (HCV E1 315), P2 (HCV E2517), P3 (HCV E2412) was tested by ELISA where (7G9) gave the highest readings (0.969),(0.330) and (0.390) respectively .
They were also tested against HCV positive samples and negative samples. The monoclonal antibody (7G9) giving the highest value also.
Depending on all these evaluations, the monoclonal antibody (7G9) was selected for the rest of the work accomplished in this study.
Further characterization was done for the monoclonal antibody (7G9) to determine the molecular weight by SDS-PAGE and the western blot transfer method, where E1 was identified at 31 kDa and E2 at 63 kDa.
The specificity of the monoclonal antibody was tested showing no reaction obtained with Brucella abortus,Salmonella typhiand HBV using ELISA .
The sensitivity was also tested by coating serial dilutions of the peptide (HCV a.a 315-323) E1 antigen to the wells using ELISA which was detected till 2.00 µg/ml of the coated antigen.
The ELISA and the Dot-ELISA were optimized to detect HCV infected samples where ELISA had a sensitivity of 80.0% detecting samples , a specificity of96 % and an efficiency of 82.2% and Dot-ELISA had a sensitivity of 76.8% detecting samples and a specificity of88.0 % and an efficiency of 78.5%.
The OD readings obtained by ELISA to detect HCV E1/E2 antigens were compared with the viral load measured by PCR.The results reflected absence of correlation between
In this study, revival and propagation of cell lines of each of the four hybrids (7G9),(6D11),(3F6),(6E4) producing the monoclonal antibodies targetingHCV E1/E2 envelope proteins was accomplished.
All four supernatants of these hybrids showed positive results for the presence of the monoclonal antibodies targeting HCV E1/E2 envelope proteins by ELISA. The hybrid (7G9 ) showing the highest OD reading (2.6) while the positive control was (1.7) and the negative control was (0.4).
The supernatants were purified by the ammonium sulphate precipitation method to obtain the monoclonal antibodies.
The monoclonal antibodies (7G9),(6D11),(3F6),(6E4) were characterized by determining their isotypeswhich were found to be IgM/Kappa chain for all.
The protein content of each of them was also determined where (7G9) was 9.5mg/ml, (6D11) was 9.75mg/ml, (3F6) was 10.5mg/ml and (6E4) was 8.0 mg/ml.
The reactivity of the four monoclonal antibodies against the peptide antigens P1 (HCV E1 315), P2 (HCV E2517), P3 (HCV E2412) was tested by ELISA where (7G9) gave the highest readings (0.969),(0.330) and (0.390) respectively .
They were also tested against HCV positive samples and negative samples. The monoclonal antibody (7G9) giving the highest value also.
Depending on all these evaluations, the monoclonal antibody (7G9) was selected for the rest of the work accomplished in this study.
Further characterization was done for the monoclonal antibody (7G9) to determine the molecular weight by SDS-PAGE and the western blot transfer method, where E1 was identified at 31 kDa and E2 at 63 kDa.
The specificity of the monoclonal antibody was tested showing no reaction obtained with Brucella abortus,Salmonella typhiand HBV using ELISA .
The sensitivity was also tested by coating serial dilutions of the peptide (HCV a.a 315-323) E1 antigen to the wells using ELISA which was detected till 2.00 µg/ml of the coated antigen.
The ELISA and the Dot-ELISA were optimized to detect HCV infected samples where ELISA had a sensitivity of 80.0% detecting samples , a specificity of96 % and an efficiency of 82.2% and Dot-ELISA had a sensitivity of 76.8% detecting samples and a specificity of88.0 % and an efficiency of 78.5%.
The OD readings obtained by ELISA to detect HCV E1/E2 antigens were compared with the viral load measured by PCR.The results reflected absence of correlation between
Other data
| Title | Diagnostic and Biochemical Characterization of Hepatitis C Virus Envelope Protein Using Specific Monoclonal Antibodies | Other Titles | التشخيص والوصف الكيميائى الحيوى للغلاف البروتينى لفيروس الألتهاب الكبدى الوبائى سي باستخدام أجسام مضادة وحيدة النسيلة | Authors | Iman Ali Mohamed El Demerdash Touni | Issue Date | 2017 |
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