Efficient generation of recombinant anti-HER2 scFv with high yield and purity using a simple method

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Human epithelial growth factor receptor 2 (HER2) is a key target in breast cancer diagnostics and therapeutics.
• Developing efficient and sensitive methods for HER2 detection is crucial for personalized medicine.

Purpose of the Study:

• To develop a method for producing a soluble single-chain fragment variable (scFv) antibody targeting HER2 in Escherichia coli.
• To optimize scFv production for high yield, purity, and HER2-binding activity.
• To evaluate the utility of the developed scFv as a diagnostic probe for HER2-positive breast cancer.

Main Methods:

• Optimized orientations of variable heavy (VH) and variable light (VL) domains and His-tag for scFv production in E. coli.
• Purification of the HL-His type antibody.
• Determination of purity and limit of detection.
• Labeling of HER2-positive and HER2-negative cell lines with fluorescent dye-conjugated scFv.

Main Results:

• Identified HL-His type antibody with highest HER2-binding activity.
• Achieved high yield (40.7 mg/L) and >99% purity of scFv.
• Determined a low limit of detection (2.9 ng).
• Demonstrated significantly higher signal-to-background ratio in HER2-positive cell lines using fluorescent scFv probes.
• Streamlined production process completed in 4-5 days with stable freeze storage.

Conclusions:

• The developed scFv antibody production method is efficient, yielding a highly pure and sensitive product.
• Fluorescent scFv probes show significant potential for accurate HER2-positive breast cancer diagnostics.
• The rapid, scalable, and stable nature of the scFv product supports its practical application in clinical settings.