Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs

Hedegaard, Chris J.; Strube, Mikael L.; Hansen, Marie B.; Lindved, Bodil K.; Lihme, Allan; Boye, Mette; Heegaard, Peter M. H.

Competitive ELISA for determination of binding of antibodies to specific antigens: wells were coated with DOC antigen extracts in 0.1 M sodium carbonate buffer pH 9.6 at 3.3 μg/ml (E. coli extract) or 4.8 μg/ml (Salmonella diarizonae). After blocking, swine immunoglobulins and horse radish peroxidase (HRP)-conjugated antibodies, either goat polyclonal anti-E. coli (18-511-245057) or rabbit polyclonal anti-salmonella (18-511-245055) (both Genway Biotech. Inc., Hölzel Diagnostika, Cologne, Germany) were diluted in deionised water with 2.5% casein Hammerstein (VWR-Bie & Berntsen A/S) pH 7 (2.5 μg/ml (anti-salmonella; 18-511-245055), 5 μg/ml (anti-E. coli; 18-511-245057)) and incubated for 1 hr. The plates were then washed, developed and read as described above. Each plate held several controls with no swine immunoglobulins added in order to define the uninhibited OD in the remaining sample. From this the % inhibition in a sample was defined as 100 À ODsample ODcontrol à 100. Whole-cell ELISA for demonstration of antibody binding to bacterial surfaces: wells were coated with fixed bacteria in 0.1 M sodium carbonate buffer pH 9.6 (final OD 546 = 0.25). After blocking, swine plasma IgG was added in 2-fold dilution series from 10 to 0.02 mg/ml. After 1 hour of incubation and 3 washes in PBS-T, detection antibody (HRP-conjugated rabbit antiporcine Ig (DAKO, Glostrup, Denmark) diluted 1/7000 or HRP-conjugated goat anti-pig IgG (GGHL-5P; ICL, SMS Gruppen; Rungsted, Denmark) diluted 1/2000) was added and incubated for 1 hour. After washing, plates were developed and measured as described above.