Stepwise PEG Precipitation Coupled with Hydrophobic Interaction Chromatography Enables the Production of Highly Specific Anti-Ag85B IgY for Tuberculosis Immunodiagnostics

Purpose: Reliable antigen detection in tuberculosis (TB) immunoassays requires antibodies with high specificity and minimal background interference. Chicken egg yolk immunoglobulin Y (IgY) is a scalable and cost-effective antibody source; however, crude yolk extracts often show reduced specificity due to non-target proteins. Although antigen 85B (Ag85B) is a well-established Mycobacterium tuberculosis antigen, the generation and purification of Ag85B-specific IgY from egg yolk have not yet been reported. This study established a purification workflow to obtain high-purity Ag85B-specific IgY from chicken egg yolk. Methods: Chickens were immunized with recombinant Ag85B, and antibody development was monitored using agar gel precipitation tests (AGPT) with serum collected before and after immunization. Dot blot analysis verified the transfer of anti-Ag85B antibodies from serum to egg yolk prior to IgY extraction. IgY was extracted using either stepwise polyethylene glycol (PEG) precipitation or a NaCl-based water-dilution method, followed by hydrophobic interaction chromatography and centrifugal ultrafiltration. Results: SDS-PAGE demonstrated progressive IgY enrichment and contaminant removal, with PEG extraction yielding higher purity than NaCl-based extraction. Western blot analysis confirmed a distinct immunoreactive band for Ag85B-His with no cross-reactivity against the non-target M. tuberculosis antigen MPT64. Furthermore, a prototype membrane-based strip assay using purified IgY produced clear signals in both the test and control zones when tested with Ag85B (10 μg/mL), indicating retained antigen-recognition capability in a preliminary membrane-based proof-of-concept format. Conclusion: These findings demonstrate that systematic purification improves IgY specificity and supports the preliminary development of membrane-based proof-of-concept immunodetection systems using Ag85B-specific IgY. A flowchart of IgY extraction and assay development for Ag85B antigen with purification results.The process begins with immunization and antibody development using recombinant Ag85B antigen in chickens. Monitoring is done via Agar Gel Precipitation Test (AGPT) and Dot Blot, showing serum transfer to yolk. Comparative IgY extraction methods include Method A: PEG Precipitation and Method B: NaCl-based Water Dilution, followed by hydrophobic interaction chromatography and centrifugal ultrafiltration, resulting in enriched anti-Ag85B IgY. The result section shows purity and specificity via SDS-PAGE and Western Blot, indicating PEG yields highly purified IgY with high specificity for Ag85B. The proof of concept assay uses a prototype membrane-based strip assay with control and test zones, confirming retained antigen recognition. The conclusion states that systematic purification improves IgY specificity, supporting scalable TB immunodiagnostic platforms with minimal background interference.