A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging

Area of Science:

• Bioconjugation chemistry
• Bioimaging techniques
• Immunological assays

Background:

• Luminescent lanthanide chelates are crucial for time-gated luminescence (TGL) bioimaging.
• Directly labeling antibodies with lanthanide-binding ligands presents challenges in controlling ligand/protein ratios and preserving antibody function.
• Existing methods often compromise antibody affinity and avidity during labeling.

Purpose of the Study:

• To develop a novel indirect detection reagent for antibody labeling with luminescence.
• To circumvent the difficulties associated with direct antibody labeling.
• To enable efficient and controlled luminescence labeling of antibodies for bioimaging applications.

Main Methods:

• Development of a recombinant fusion protein, Linker-Protein G (LPG), with available lysine residues for labeling.
• Attachment of succinimide-activated lanthanide chelating ligands to lysine residues in LPG and Protein G.
• Comparison of ligand incorporation and conjugation efficiency between LPG and Protein G.
• Demonstration of luminescence labeling of cell-specific antibodies and application in TGL bioimaging of parasitic cysts and oocysts.

Main Results:

• LPG showed significantly higher and more efficient incorporation of lanthanide chelating ligands compared to Protein G alone.
• The presence of lysine residues in the linker region of LPG facilitated superior ligand incorporation.
• The developed Luminescence-Activating (LA-) complexes enabled rapid, uncomplicated luminescence labeling of antibodies.
• Successful application in TGL bioimaging of Giardia cysts and Cryptosporidium oocysts was demonstrated.

Conclusions:

• The indirect labeling approach using the LPG fusion protein offers an efficient strategy for antibody conjugation with lanthanide chelates.
• This method overcomes limitations of direct labeling, preserving antibody integrity and enabling controlled luminescence.
• The developed LA-conjugates provide a versatile tool for sensitive detection and bioimaging in various biological applications, including pathogen identification.