A Sett1, B B Borthakur2, U Bora3,4. 1. Bioengineering Research Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India. 2. Department of Surgical Oncology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, 781016, India. 3. Bioengineering Research Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India. ubora@iitg.ernet.in. 4. Mugagen Laboratories Pvt. Ltd., Technology Incubation Centre, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India. ubora@iitg.ernet.in.
Abstract
BACKGROUND: Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas. METHODS: We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate was also verified with the tissue samples of breast cancer patients by immunohistochemistry process. RESULTS: Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K d = 6.33 ± 0.86 nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 490 nm wavelength) in the dynamic range of 100-700 nM ECD_Apt1 with a detection limit of 12.5 ± 2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes. CONCLUSIONS: Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 could be an effective low-cost alternative to conventional anti-Her2 antibody in solid phase immunoassays for cancer diagnosis and related applications.
BACKGROUND:Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas. METHODS: We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate was also verified with the tissue samples of breast cancerpatients by immunohistochemistry process. RESULTS: Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K d = 6.33 ± 0.86 nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 490 nm wavelength) in the dynamic range of 100-700 nM ECD_Apt1 with a detection limit of 12.5 ± 2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes. CONCLUSIONS: Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 could be an effective low-cost alternative to conventional anti-Her2 antibody in solid phase immunoassays for cancer diagnosis and related applications.
Entities:
Keywords:
DNA aptamers; HER2; Immunoassays; Immunohistochemistry
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