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.

For immunoblotting, separated proteins were electroblotted from SDS PAGE gels onto nitrocellulose membranes (Millipore, Copenhagen, Denmark) in a Mini Trans-Blot transfer cell including a cooling unit (Bio-Rad) with 12.5 mM Tris, 96 mM Glycine (both from Sigma-Aldrich, Copenhagen, Denmark), 20% Ethanol (VWR-Bie & Berntsen A/S, Herlev, Denmark) pH 8.4 for 1 hour at 150 mA constant current. All subsequent operations were performed at room temperature with shaking. Next, the membranes were blocked in 50 mM Tris, 250 mM NaCl (TBS) with 2% Tween 20 at pH 8.6 for 5 minutes; followed by 3x10 min washes in TBS-T (TBS with 0.1% Tween 20 (Merck, Hellerup, Denmark)). Then, membranes were incubated with polyclonal biotinylated rabbit anti-Pig F(ab) 2 IgG (LSBio, Copenhagen, Denmark) diluted 1:10.000 in TBS-T for 1 hour followed by 3x10 min washes in TBS-T before the last incubation with alkaline phosphatase-conjugated streptavidin (DAKO, Glostrup, Denmark) 1:3000 in TBS-T (1 hour). After 3x50 ml washes in TBS-T, blots were developed with 4-nitro-blue-tetrazolium/5-bromo-4-chlor o-3-indolyl-phosphate (NBT/BCIP) tablets (Roche, Hvidovre, Denmark) following the manufacturer's instructions, terminating colour development by washing the blot with several changes of MilliQ water.