Development of a novel method for generating Q-bodies using tyrosinase-mediated HA-tag labeling

Area of Science:

• Biotechnology
• Immunology
• Analytical Chemistry

Background:

• Quenchbodies (Q-bodies) are fluorescent antibodies utilizing fluorescence quenching/de-quenching upon antigen binding.
• Existing Q-body generation methods have limitations in versatility and color range.

Purpose of the Study:

• To develop a novel and versatile Q-body generation strategy.
• To expand the color palette of Q-bodies through site-specific dye conjugation.
• To create a Q-body targeting programmed cell death-ligand 1 (PDL1).

Main Methods:

• Engineered a single-chain variable fragment (scFv) against PDL1 with an N-terminal hemagglutinin (HA)-tag.
• Expressed the scFv in Escherichia coli for high yield and purity.
• Utilized tyrosinase-mediated site-specific conjugation of hydrazide-functionalized dyes to the HA-tag.

Main Results:

• Successfully generated Q-bodies with four different emission wavelengths.
• All generated Q-bodies showed antigen concentration-dependent fluorescence enhancement.
• The TAMRA-labeled Q-body achieved a high signal-to-background ratio and a limit of detection of 0.51 ± 0.01 μg/mL for PDL1.

Conclusions:

• The HA-tag-mediated Q-body generation is a robust and versatile method.
• This strategy allows for broad dye compatibility in Q-body production.
• Enables sensitive and multiplexed fluorescent immunoassays for various targets.