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Optimization of HER2-based and cell-based ELISA for detection of trastuzumab biosimilar
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Maryam Ahmadzadeh    , Farzaneh Farshdari , Leila Nematollahi , Shayan Maleknia , Elham Mohit |
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Abstract: (3392 Views) |
| Introduction: ELISA is a sensitive, specific, reproducible and fast method to quantify the biological activity of antibodies. Trastuzumab is a humanized monoclonal antibody against HER2 receptors which prevents the initiation of downstream signaling pathway. Trastuzumab can be used as a positive control in the ELISA experiments for anti-HER2 antibodies. Additionaly, insufficient washing and blocking can be the cause of background in the ELISA experiment that is necessary to be optimized. As we do not want to waste antigen, it is also important to determine the last dilution of antigen that can give good titration curve for trastuzumab. The aim of this study was to optimize ELISA method for trastuzumab biosimilar (AryoTrust™, Aryogen pharmed).
Materials and Methods: In this study, different variables including the number of washing cycle, the blocking agent and antigen concentration in HER2-based ELISA and the type of HER2 negative cell line and the blocking agent in cell-based ELISA were studied.
Results: It was demonstrated that 5 times washing between different steps of HER2-based ELISA causes significant lower non-specific background as compared to 3 times washing. Moreover, the nonspecific binding was significantly lower in the presence of % 5 skim milk as blocking agent as compared to BSA %1 or BSA %3. In addition, the lowest HER2 concentration which gives good titration curve for trastuzumab was 0.1 μg/ml. In cell-based ELISA experiment, it was demonstrated that the use of MDA-MB-231 as negative HER2 cell line caused significant lower background than MCF-7. Furthermore, BSA 3% was chosen as proper blocking agent.
Conclusion: The results of this study can be used for development of HER2 and cell-based ELISA for anti HER2 antibodies and its fragments. |
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| Keywords: Enzyme-Linked Immunosorbent Assay (ELISA), Biological Activity, Trastuzumab, Antibodies, Optimization. |
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Full-Text [PDF 1171 kb]
(810 Downloads)
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Type of Study: Research |
Subject:
General
Received: 2019/07/1 | Accepted: 2019/09/25 | Published: 2020/03/15
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