ABSTRACT

Background and Aim: Mercury (Hg) is a persistent environmental toxin that poses significant risks to animal and human health through contaminated feed, water, and ecosystems. Conventional instrumental methods for Hg detection are laboratory-intensive, while immunochemical approaches require highly specific antibodies. Hg ions are poorly immunogenic and therefore require hapten–carrier conjugation. This proof-of-concept study aimed to evaluate the feasibility of generating functional Hg-reactive immunoglobulin Y (IgY) antibodies in laying hens using Hg chloride (HgCl2)–protein conjugates, offering an avian alternative to mammalian antibody production for veterinary toxicology and environmental monitoring applications. 

Materials and Methods: HgCl₂ was conjugated to keyhole limpet hemocyanin (KLH) using 3-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as crosslinker and emulsified with Freund’s adjuvants. Two Hy-Line Brown laying hens received subcutaneous immunizations (primary dose: 100 μg antigen in complete Freund’s adjuvant [CFA]; booster doses: 50 μg antigen in incomplete Freund’s adjuvant on days 14 and 28). Due to irregular egg production, serum was used as the antibody source. Antibody reactivity was assessed by indirect enzyme-linked immunosorbent assay (ELISA) using HgCl2–bovine serum albumin (BSA) as coating antigen. IgY was purified by caprylic acid and ammonium sulfate precipitation and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The study was conducted under strict ethical approval (No. 2403/FapetUN/ETIK/2024) and biosafety protocols, with a minimal cohort size justified by constraints on Hg handling. 

Results: In the single hen that completed the full immunization protocol, post-immunization serum (day 42) showed strong reactivity at 10 μg/mL HgCl2–BSA coating. Optical density at 450 nm (OD₄₅₀) ranged from 0.42 to 0.67 compared with pre-immune baseline values below 0.22, producing a fold increase over pre-immune serum of 1.93–11.41× (9 of 15 conditions >5×; 6 of 15 conditions >8×). Intra-assay coefficient of variation remained below 10% (mean 4.27%). Purified IgY displayed intact heavy chain (~65 kDa) and light chain (~25 kDa) bands on reducing SDS-PAGE, confirming successful enrichment. 

Conclusion: This preliminary observation demonstrates successful induction of Hg-reactive IgY antibodies in a laying hen using a hapten–carrier immunization strategy. The robust ELISA discrimination supports further development of IgY-based tools for Hg screening. Future studies must include ≥3 biological replicates, hapten–density quantification, specificity controls, and competitive assay formats for proper validation. 

Keywords: avian antibodies, environmental Hg detection, hapten–carrier conjugate, immunoglobulin Y, laying hens, Hg IgY, Hg-reactive antibodies, veterinary toxicology.