Flow immunochemical bio-recognition detection for the determination of interleukin-10 in cell samples

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• Interleukin-10 (IL-10) is a critical cytokine with diverse roles in immune regulation.
• Accurate and sensitive detection of IL-10 is essential for research and diagnostics.
• Existing methods for IL-10 determination may have limitations in speed or sensitivity.

Purpose of the Study:

• To develop and optimize on- and off-line heterogeneous non-competitive flow immunoassays for Interleukin-10 (IL-10) determination.
• To compare the performance characteristics, including detection limits and sample throughput, of the on-line and off-line assay formats.
• To validate the assays using a spiked cell medium matrix and confirm IL-10 identification using MALDI-TOF MS.

Main Methods:

• Heterogeneous non-competitive flow immunoassays utilizing fluorescently labeled anti-IL-10 antibodies.
• On-line (in-column) and off-line (test tube) incubation modes for antibody-antigen complex formation.
• Trapping of unbound antibodies on an immobilized IL-10 column and detection of eluted complexes via fluorescence.
• Optimization of assay parameters including affinity support, flow rate, buffer composition, pH, and antibody-antigen association.
• Matrix validation using spiked cell medium and IL-10 identification by MALDI-TOF MS.

Main Results:

• The off-line incubation mode achieved a lower IL-10 detection limit of 8 fmol compared to 40 fmol for the on-line mode in spiked cell medium.
• Sample throughput was higher in the off-line mode (40 samples/hour) than in the on-line mode (26 samples/hour).
• Both assay formats demonstrated successful IL-10 identification when coupled with MALDI-TOF MS.

Conclusions:

• Both on- and off-line flow immunoassays provide sensitive and reliable methods for IL-10 determination.
• The off-line assay format offers superior sensitivity, while the on-line format provides higher sample throughput.
• These developed immunoassays are valuable tools for quantifying IL-10 in biological samples, with potential applications in research and diagnostics.