Protein A/G-based microfluidic platform for preparative microseparation of IgG from human serum

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Immunodiagnostics

Background:

• Immunoglobulin G (IgG) antibodies are key targets in serological assays, crucial for detecting past infections or vaccination responses.
• Existing serological assays face challenges with matrix-derived background and variability, complicating diagnostic interpretation.
• Current IgG purification methods are often not compatible with miniaturized diagnostic systems, hindering advancements in point-of-care testing.

Purpose of the Study:

• To develop a miniaturized, automation-ready pre-analytical module for efficient IgG antibody separation.
• To address the limitations of existing purification tools in the context of miniaturized detection systems.
• To improve the signal-to-noise ratio and reduce variability in serological assays.

Main Methods:

• Development of an open-channel PET Lab-on-a-Foil cassette with protein A/G-functionalized microchannels for reversible IgG capture.
• Implementation of an automated bind-wash-elute sequence using pH-triggered IgG release.
• Validation using Enzyme-Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance (SPR) with SARS-CoV-2 serology as a model.

Main Results:

• The developed cassette yielded approximately 30 μL of IgG-enriched eluate per run.
• IgG-enriched fractions showed significantly reduced levels of C-reactive protein and minimal carryover of free protein A/G.
• The assay detected a 7.6-fold higher antigen-specific signal for IgG anti-SARS-CoV-2 nucleoprotein in positive versus negative serum eluates.
• Nonspecific adsorption, measured by SPR, decreased by 80-90% after pre-treatment, confirming reduced background.

Conclusions:

• The integrated IgG capture and elution platform in a disposable PET cassette effectively lowers blank signal and variance near diagnostic cut-offs.
• This technology is compatible with downstream detection systems like ELISA and SPR.
• The developed module offers a practical approach for automation-compatible point-of-care immunodiagnostics, enhancing diagnostic precision.