Agarose

A modified version of the Jerne haemolytic plaque assay^{57 (link)} was used. Spleens were harvested 5 days after immunization and single cell suspensions prepared in Hank’s balanced salt solution. One hundred μl of appropriately diluted spleen cells, 25 μl 10% SRBC suspension, and 25 μl guinea pig serum (as a source of complement, diluted 1:10) (National Veterinary Institute) were added to 300 μl 0.5% agarose at 45 °C (50% Seaplaque agarose (low melting point, Amersham) and 50% agarose (United States Biochemical)). The mixture was immediately spread on a microscope slide and incubated in a humid chamber for 3 h at 37 °C. Hank’s balanced salt solution were was used for all dilutions. Triplicate samples were counted “blindly”.

Trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (CCA), acetonitrile (ACN) and dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO, USA). Immobiline drystrips, ammonium persulfate, urea, agarose, glycerol, bromophenol blue, iodoacetamide (IAA), silver nitrate, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane (CHAPS), and N, N, N’, N’-tetramethylethylenediamine (TEMED) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Acrylamide, Bis, Tris, glycine, SDS and SDS-PAGE protein standards were from Bio-Rad (Hercules, CA).