Veterinary world · 2026
Formation and preliminary characterization of mercury-reactive immunoglobulin Y antibodies in laying hens using HgCl2-protein hapten conjugates.
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Wariata Wayan, Sriasih Made, Rosyidi Anwar, Depamede Sulaiman Ngongu
Аннотация
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.
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.