PURIFICATION AND N-TERMINAL AMINO-ACID SEQUENCING OF ECHINOCOCCUS-GRANULOSUS ANTIGEN-5

Antigen 5, a major parasite-derived lipoprotein of unilocular hydatid (Echinococcus granulosus) cyst fluid comprises subunits of 56-70 kDa w hich dissociate on disulphide bound reduction to two subunits of appro ximately 25 and 38 kDa on SDS-PAGE. The 38 kDa component is recognized as a potentially important diagnostic marker for cystic hydatid disea se. Single dimensional SDS-PAGE, two dimensional (2-D) gel electrophor esis, modified '2-D' gel electrophoresis, immunoaffinity-purification and HLPC were employed to purify antigen 5. Subsequent N-terminal sequ encing suggested that antigen 5 isoforms exist due to the identificati on of a single amino acid sequence with alternative residues at some p ositions. An 18 amino acid consensus N-terminal sequence was derived f or antigen 5 as a result of comparing the sequences obtained by the di fferent fractionation methods. No homology was revealed to any previou sly identified protein including putative antigen 5 amino acid sequenc es. A synthetic peptide, mimicking the N-terminal consensus sequence, did not bind with patient sera (confirmed positive to antigen 5) or an anti-antigen 5 MoAb. Protein sequences (16 residues compared) for the 38 kDa subunit from sheep and horse (representing different strains o f E. granulosus) cyst fluids were very similar except for differences at three positions. 2-D and modified '2-D' gel electrophoresis, in com bination with immunoblot analysis, indicated the presence of more than one protein in the 38 kDa legion of SCF capable of binding the anti-a ntigen 5 MoAb.