Generation and Validation of Antibodies Targeting AD1 Containing Isoform of Alternatively Spliced TNC

Ali S Alhareth^1, , Musaad A Alsulaiman², Abo baker I Alshomrani², Waleed A Alyami³ and Hamad M Harthi⁴

¹Laboratory and blood bank (Iman Hospital), Riyadh Cluster 1, Ministry of Health, Riyadh, KSA

²Department of Public Health, Ministry of Health, Riyadh, KSA

³Riyadh Health Affairs, Ministry of Health, Riyadh, KSA

⁴Emergency and Disaster Management, Ministry of Health, Najran, KSA

Cite this paper

Ali S Alhareth, Musaad A Alsulaiman, Abo baker I Alshomrani, Waleed A Alyami and Hamad M Harthi. Generation and Validation of Antibodies Targeting AD1 Containing Isoform of Alternatively Spliced TNC. American Journal of Medical and Biological Research. 2019; 7(1):24-28. doi: 10.12691/AJMBR-7-1-5

Abstract

Tenascin-C (TNC), a matricellular protein. TNC-AD1 isoform is molecular weight (MW) isoform has been shown to be over-expressed in high grade carcinoma hormone insensitive breast cancers in younger women. The aim of this study was to generate antibodies targeting the AD1 domain for use in tissue and functional studies. Bioinformatics analysis was preformed to select sequences within the AD1 domain for targeting with anti-peptide antibodies. Subsequently, the effect of siRNA-mediated knockdown of AD1-containing isoforms of TNC was carried out to analyse to validate the antibody specificity. In addition, the successful antibody for the detection of TNC-AD1 specify was also tested in breast cancer tissue. Immunocytochemistry and Immunohistochemistry analysis showed specific reactivity for the generated antibody with staining found to be cytoplasmic in cell lines and both the cytoplasm and ECM in breast cancer tissues; whereas western blot analysis showed no immunoreactivity detected for TNC-AD1 expression using the anti-AD1 antibodies. In conclusion, the generated antibody against TNC-AD1 was successfully recognised TNC-AD1 may serve as a specific tool for further functional studies of the pathological role of TNC-AD1 in breast cancer.

Keywords

TNC, AD1

Copyright

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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