Fluorescent labeling of antibody fragments using split GFP.

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Antibody fragments, like single chain Fvs (scFvs), are valuable tools in biotechnology.
• Traditional labeling of scFvs often requires small peptide tags due to the absence of the Fc portion.
• Existing labeling methods can sometimes affect scFv expression or functionality.

Purpose of the Study:

• To develop an efficient and non-perturbing method for fluorescently labeling scFvs.
• To demonstrate the utility of the split green fluorescent protein (GFP) system for scFv labeling.
• To enable functional characterization of scFvs directly in crude samples.

Main Methods:

• Fusion of a 13 amino acid GFP tag (GFP11) to the C terminus of scFvs.
• Utilizing the split GFP system for fluorescent labeling of scFvs.
• Assessing scFv functionality using fluorescence-linked immunosorbent assays, flow cytometry, and yeast display.

Main Results:

• The GFP11 tag did not affect scFv expression or functionality.
• Effective functional fluorescent labeling was achieved across various assays.
• The split GFP system allowed for concentration and affinity estimation of scFvs in crude samples without purification.

Conclusions:

• The split GFP system provides an efficient method for fluorescently labeling scFvs.
• This technique is non-perturbing and allows for functional assays and quantification.
• The system holds significant potential for antibody engineering and in vitro display applications.