| Autor: |
Wei E; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda Maryland 20892, United States., Bou-Nader C; Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, United States., Perry ML; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda Maryland 20892, United States., Fattah R; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda Maryland 20892, United States., Zhang J; Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, United States., Leppla SH; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda Maryland 20892, United States., Bothra A; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda Maryland 20892, United States. |
| Abstrakt: |
Diagnosis of infectious agents is increasingly done by the detection of unique nucleic acid sequences, typically using methods such as PCR that specifically amplify these sequences. A largely neglected alternative approach is to use antibodies that recognize nucleic acids. The unique monoclonal antibody S9.6 recognizes DNA-RNA hybrids in a largely sequence-independent manner. S9.6 has been used in several cases for the analysis of nucleic acids. Extending our recent determination of the structure of S9.6 Fab bound to a DNA-RNA hybrid, we have developed reagents and methods for the sensitive detection of specific DNA and RNA sequences. To facilitate the use in diagnostics, we conjugated the S9.6 Fab to the highly active and well-characterized reporter enzyme human-secreted embryonic alkaline phosphatase (SEAP). Two approaches were utilized for conjugation. The first used sortase A (SrtA), which generates a covalent peptide bond between short amino acid sequences added to recombinantly produced S9.6 Fab and SEAP. The second approach was to genetically fuse the S9.6 Fab and SEAP so that the two are produced as a single molecule. Using these two antibody-SEAP proteins, we developed a simplified ELISA format for the identification of synthetic DNA-RNA hybrids, which can be optimized for detecting nucleic acids of pathogens, as well as for other applications. We successfully used this immunosorbent assay, HC-S, to identify DNA-RNA hybrids in solution with high specificity and sensitivity. |
