MECHANICAL ISOLATION AND CHARACTERIZATION OF ANTIGENS FROM ADULT ANGUILLICOLA-CRASSUS

The serum Ig response of the European eel (Anguilla anguilla) to the b lood feeding nematode Anguillicola crassus was studied. When tested by ELISA in a previous study, it was found necessary to purify the adult worm antigens in order to reduce the unwanted high background values. In the present study an alternative method is described and evaluated . Instead of using biochemical methods, adult worms were simply dissec ted and antigens prepared from the cuticle, female and male gonads, in testinal wall and the intestinal contents. The specific antibody level s in pooled sera from eels that were (1) non-infected; (2) naturally i nfected and (3) immunized with whole-worm extract, were tested by ELIS A with these antigens and compared with the reaction to adult whole-wo rm homogenate (AWWH) and excretory-secretory (ES) antigens. In additio n, the antigen preparations were characterized by sodium dodecyl polya crylamide-gel electrophoresis (SDS-PAGE). The Western blotting techniq ue (WB) was used to determine their recognition by antibodies in eel s era. The results obtained support the idea that remnants of eel blood present in the gastrointestinal tract of the adult worm generate high background values in the ELISA. However, when we characterized the ant igen preparations that reacted similarly with low background values in the ELISA (i.e. the cuticle and the gonads), major differences were s een both in the protein banding pattern and between the bands that wer e recognized by the antibodies following WB. For example, the cuticle contained a distinct specific band with an approximate molecular weigh t of 45 kDa that was not seen in preparations of the gonads. It can be concluded that the cuticle of A. crassus contains specific antigens w hich can easily be isolated. The described method for antigen processi ng is simple and the isolated cuticle antigens proved to be specific i n an ELISA measuring the antibody response to A. crassus in the Europe an eel.