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Updated: Mar 28, 2026
Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells
Published on: May 9, 2025
Development and evaluation of a single domain antibody against human epidermal growth factor receptor (EGFR)
Shiva Ahdi Khosroshahi1, Safar Farajnia2, Mehdi Ghiamirad3
1Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract:
Epidermal growth factor receptor (EGFR) plays an important role in cell growth, multiplication and differentiation. Over expression of EGFR is associated with carcinogenesis and seen in variety of cancers. Anti-EGFR monoclonal antibodies can block EGFR downstream signaling pathway resulting in inhibition of uncontrolled cell proliferation. Antibody fragments have a variety of advantages. In comparison to full length antibodies they have smaller size and therefor exhibit better tumor penetration ability. The aim of this study was to prepare a single domain antibody to target extracellular domain of EGFR. mRNA was extracted from C225 hybridoma cells producing anti-EGFR antibody and subjected to reverse transcription reaction (RT-PCR) to obtain cDNA molecules encoding VH domain of mAb C225. The cDNA encoded VH domain was in frame introduced into the pET-22b(+) vector and expressed in BL21 (DE3) bacterial cells. The resultant antibody was purified via Ni- NTA column and its reactivity was assessed by ELISA and western blot techniques using A431 cell lysate. Analysis by ELISA revealed that this single domain antibody was able to bind EGFR on A431cells. This result was further confirmed by western blotting. In conclusion, the results of this study indicated that single domain antibody can identify and bind to EGFR of A431 carcinoma cells. This recombinant fragment antibody would potentially be used for targeting of cancer cells with high EGFR expression.
Insights
Researchers developed a single domain antibody targeting the epidermal growth factor receptor (EGFR). This antibody fragment effectively binds to EGFR on cancer cells, showing potential for targeted cancer therapy.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Epidermal growth factor receptor (EGFR) is crucial for cell functions and its overexpression is linked to various cancers.
- Monoclonal antibodies targeting EGFR inhibit cancer cell proliferation by blocking downstream signaling.
- Antibody fragments offer advantages like smaller size and enhanced tumor penetration compared to full antibodies.
Purpose of the Study:
- To engineer a single domain antibody fragment targeting the extracellular domain of EGFR.
- To assess the binding and reactivity of the developed antibody fragment to EGFR.
Main Methods:
- Obtained cDNA encoding the VH domain of an anti-EGFR antibody (mAb C225) from hybridoma cells via RT-PCR.
- Expressed the VH domain in bacterial cells (BL21 DE3) using a pET-22b(+) vector.
- Purified the antibody fragment using Ni-NTA chromatography and evaluated its reactivity via ELISA and Western blot.
Main Results:
- ELISA confirmed the single domain antibody's ability to bind EGFR on A431 cells.
- Western blot analysis further validated the binding of the antibody fragment to EGFR.
- The study successfully generated a functional single domain antibody targeting EGFR.
Conclusions:
- The developed single domain antibody fragment specifically identifies and binds to EGFR on A431 carcinoma cells.
- This recombinant antibody fragment holds potential for targeted therapy in cancers with high EGFR expression.
- Single domain antibodies represent a promising alternative for cancer targeting strategies.
