Partial purification and characterization of protection-inducing antigens from the muscle larva of Trichinella spiralis by molecular sizing chromatography and preparative flatbed isoelectric focusing

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Abstract
The soluble portion of a large particle fraction which was derived from the muscle larva of T. spiralis was subjected to molecular sizing column chromatography using Sephacryl S-200. Five major peaks of 280 nm absorbing material were obtained. Analysis by immunoelectrophoresis revealed that each peak contained antigens, with the majority of them occurring in peaks 3,4 and 5. Preliminary studies indicated that peak 4 (mol. wt range 20 000-10 000) contained protection-inducing antigens. Crossed-immunoelectrophoretic and single-dimension electrophoretic analysis of peak 4 revealed a minimum of 10 antigens, while analytical isoelectric focusing demonstrated the presence of proteins with widely different pl, ranging from 4·0 to 9·0. Peak 4 was fractionated by preparative flatbed isoeletric focusing (PIEF) using two gradients: one from 3·5 to 9·5 and the other from 3·5 to 5·5. Fused rocket immunoelectrophoretic (FRIEP) analysis of both runs indicated that several antigens were separated from the others: one at pl 4·0 and the other at pl 9·0. The remaining antigens focused between pl 4·3 and 4·9. One hundred micrograms of whole peak 4, pl 9·0 antigen and the group of antigens at pl 4·3–4·9 were each separately injected, along with Freund's complete adjuvant, into mice. In addition, a portion of the pl 4·0 antigen was also assayed for protection. All antigenic preparations induced significant levels of protection. The pl 4·0 was further analysed on high-performance liquid chromatography (HPLC). Two sharp peaks of antigen, as detected by FRIEP, were eluted isocratically with 65% acetonitrile from a (2–18 (aliphatic) column. Both peaks of antigen showed complete cross-reactivity on FRIEP and absorbed at 220 nm. Amino acid analysis of each HPLC peak revealed no detectable differences in composition. Each peak contained a predominance of aspartic (13 mol%) and glutamic (18 mol%) acid. This antigen did not contain significant quantities of aromatic amino acids, and absorbed strongly at 206 nm. Neither the pl 4·0 or pl 9·0 antigen stained positively with the PAS reaction.