AP166P Sigma-AldrichGoat Anti-Porcine IgG Antibody, HRP conjugate

Experimental vaccination with radiation-attenuated cercariae (RAC) confers possible practical levels of resistance to challenge infection by humoral and by cellular mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG antibody from RAC vaccinated miniature pig. Two milligrams of soluble egg antigen (SEA) or schistosomal worm antigen preparation (SWAP) was fractionated using two dimensional liquid chromatography (proteome PF 2D) consisted of high performance chromatofocusing (HPCF) and high resolution reversed phase chromatography (HPRP). Of the 42 HPCF fractions of SEA or SWAP, 26 (61.9%) or 15 (35.7%) showed positive dot blot reaction with RAC vaccinated serum respectively. The dot blot positive fractions were applied to the second HPRP column. One hundred and seven out of 26 x 96 of SEA fractions and 18 out of 15 x 96 SWAP fractions reacted with RAC vaccinated serum. From the positive fractions we chose 17 of SEA and 10 of SWAP that had no reactivity with normal cercariae infected (NCI) sera and had single peak of 214 nm; and automated N-terminal amino acid sequence based on in situ Edman Reaction was conducted. Four sequences were obtained and applied to the homology search in NCBI database. A total of eight candidate genes were listed up and their cDNA clones from schistosomula stage were obtained. Two of the recombinant proteins (AAW27472.1 and AXX25883.1) showed strong reactivity with the RAC vaccinated serum but marginal with NCI serum. This protocol using proteome PF 2D could be applicable in identifying immunoreactive proteins from crude extract for the development of vaccines or for diagnostics.