Department of Periodontology School of Stomatology, Anhui Medical University, Hefei 230032, China.
To obtain egg yolk antibody in hen eggs laid by hens immunized with the protein of Porphyromonas gingivalis (Pg). To generate, purify IgY against Pg (anti-Pg-IgY) and identify its specificity.
PgATCC33277 was cultured under standard anaerobic conditions and harvested after proliferation. Then Pg was extracted by sonication until the cell pellets were shattered completely. After centrifugatiton, the supernatant was collected. Five-month-old Roman hens were immunized for egg antibody production. The antibody was inoculated intramuscularly and subcutaneously in the breast from multiple spot with 1.0 ml of a vaccine consisting of oil-adjuvant protein which was mixed with 1 ml protein of Pg and 1 ml Freund's adjuvant complete every 10 days, for 4 times. The eggs were collected after the first immunization and stored at 4°C. The anti-Pg-IgY was extracted and purified. The protein concentration was tested by bicinchoninic acid (BCA), the specificity of IgY analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), the titre of IgY and its physicochemical character were evaluated by indirect enzyme-linked immunosorbent assay.
The concentration of obtained anti-Pg-IgY was 2.05 g/L. SDS-PAGE analysis of the anti-Pg-IgY showed that the molecular weight of IgY was consistent with the theoretical value. Protein of anti-Pg-IgY appeared approximately 5 days after the first immunization, and reached the peak at 50 - 55 days. Antibody titres reached 1:100 000. Each egg produced more than 10 mg IgY, and its purification was up to 95% as well.
Layer hens immuned by Pg may provide specific IgY of high titre and high concentration. The antibody has high purity and is heat, acid and alkali-resistant.