LABELLING OF PROTEIN FRACTIONS OF FASCIOLA HEPATICA ANTIGEN WITH

Tomanek J., J. Hampl, M. Sedlacek, J. Willomitzer and E. Chroustova: Labelling of Protein Fractions of Fasciola' hepatica Antigen with U5 I. Acta vet. Bmo, 50,1981: 97-100. Gel filtration of crude Fasciola hepatica antigen on Sephadex G-I00 and G-200 yielded protein fractions that reacted with serum antibodies of F. hepatiCa infected animals. The fractions were labelled with usI by the oxidation method using chloramine T. The protein-bound radioiodine percentage was 5.4 to 23.6 per cent, depending on the quality of flukes employed for the preparation of crude antigen and on the Na 1151 employed for iodination. When tested by direct radioimmunodiffusion with positive sera from F. hepatica-infected animals and with control negative sera, the 1251-labelled protein fractions showed the same specificity as the unlabelled ones. The iodinated antigen was stable for 4 weeks, and no more than 5 and 6 per cent of unreacted radioactive iodide were found after 6 and 8 weeks of storage, respectively. Stored at 4 DC, the iodinated antigen could be used for serological reactions for 5 to 8 weeks. Diagnosis, fascioliasis, direct radioimmunodijJusion, protein-bound radioiodine percentage, stability, iodinated antigen. The diagnosis of fascioliasis by serological methods has been described by a number of investigators using differently prepared antigens with varying results. The pertinent literature was reviewed by Koch (1969) and Grelck (1976). The possibility of demonstrating the antibodies by radioimmunoassay (RIA) has not been considered to date. One of the prerequisites for successful RIA is the use of a suitable antigen. Crude antigen obtained from Fasciola hepatica homogenate can be conveniently fractionated by gel filtration on a Sephadex column of appropriate type as was demonstrated by Movsesijan and Borojevic (1973). The relatively specific protein fraction separated in this way can be labelled with an radioactive isotope and used for RIA. For practical purposes it appears most convenient to use 12111 which was employed by Williams et al. (1971) for the labelling of Schistosoma mansoni antigen. Our objective was to separate the functional fraction from crude F. hepatica antigen, to test the conditions of its labelling by the oxidation method with chloramine T and to assess the properties of the iodinated antigen. Materials and Methods The antigen was prepared from adult F. hepatica obtained from the liver of infected animals. The flukes were washed in several changes of saline, dried of filter paper and frozen at -18 DC. Mter being thawed, they were ground in a glass homogenizer, macerated in saline for 24 hours at + 4 to + 6 DC and then centrifugated for 20 min. at 12000 r. p. m. The supernatant was aspirated and used for the separation of fractions by gel filtration on a 2.5 x 39 em column of Sephadex G-l00 or an a 2.5 x 85 em column of Sephadex G-200. Elution was carried out with 0.15 molll NaCl, pH 7.0,or 0.2 molll Tris HCl buffer, pH 8.0 Fractions obtained from the individual peaks were concentrated under a cold air flow in dialysis tubes and then tested by diffusion in agar gel according to Ouchterlony using rabbit and sheep sera containing antibodies produced in response

The diagnosis of fascioliasis by serological methods has been described by a number of investigators using differently prepared antigens with varying results.The pertinent literature was reviewed by Koch (1969) and Grelck (1976).The possibility of demonstrating the antibodies by radioimmunoassay (RIA) has not been considered to date.One of the prerequisites for successful RIA is the use of a suitable antigen.Crude antigen obtained from Fasciola hepatica homogenate can be conveniently fractionated by gel filtration on a Sephadex column of appropriate type as was demonstrated by Movsesijan and Borojevic (1973).The relatively specific protein fraction separated in this way can be labelled with an radioactive isotope and used for RIA.For practical purposes it appears most convenient to use 12111 which was employed by Williams et al. (1971) for the labelling of Schistosoma mansoni antigen.
Our objective was to separate the functional fraction from crude F. hepatica antigen, to test the conditions of its labelling by the oxidation method with chloramine T and to assess the properties of the iodinated antigen.

Materials and Methods
The antigen was prepared from adult F. hepatica obtained from the liver of infected animals.The flukes were washed in several changes of saline, dried of filter paper and frozen at -18 DC.Mter being thawed, they were ground in a glass homogenizer, macerated in saline for 24 hours at + 4 to + 6 DC and then centrifugated for 20 min.at 12000 r. p. m.The supernatant was aspirated and used for the separation of fractions by gel filtration on a 2.5 x 39 em column of Sephadex G-l00 or an a 2.5 x 85 em column of Sephadex G-200.Elution was carried out with 0.15 molll NaCl, pH 7.0,or 0.2 molll Tris HCl buffer, pH 8.0 Fractions obtained from the individual peaks were concentrated under a cold air flow in dialysis tubes and then tested by diffusion in agar gel according to Ouchterlony using rabbit and sheep sera containing antibodies produced in response to infection with F. hepatica.The fractions that reacted with positive sera were pooled and the protein was determined quantitatively according to Lowry et al. (1951).These fractions were labelled with 126iodine according to McConahey and Dixon (1966).Unreacted radioactive iodide was removed by dialysis against buffered saline, pH 7.2.Checks upon the completeness of its removal were carried out by ascending paper chromatography according to Schmidt et al. (1979) and the results were evaluated by measurement in an Actigraph III, Nuclear Chicago.
Antigenic specificity of the 125I-Iabelled fractions was checked by direct radioimmunodiffusion with antibody-containing sera from F. hepatica-infected animals and with negative sera.The results were evaluated autoradiographically using a Medix Rapid R3 film, Foma, at an exposure time of 24 to 72 hours.When tested by direct radioimmunodiffusion with positive sera from F. hepatica-infected animals and control negative sera, the 125I-Iabelled antigen fractions showed the same specificity and immunoprecipitating properties as the unlabelled fractions.

Gel filtration on
The stability of the iodinated antigen as evaluated at weekly intervals proved relatively good.The antigen was stable for 4 weeks, and no more than 5 and 6 percent of unreacted radioactive iodide were found after 6 and 8 weeks of storage, respectively.Stored at 4 °C without a preservative, the iodinated antigen could be used for serological reactions for 5 to 8 weeks.Longer storage resulted in the formation of sediment and a higher rate of release of radioactive iodide.

Discussion
Crude antigen prepared from F. hepatica homogenate contains not only the functional antigen capable of reaction with antibodies produced in the serum of infected animals but also a number of non-standard substances affecting the specificity of reactions.A considerable proportion of the protein content is made up of proteins of the host from which the flukes were obtained.Fractionation and purification of the functional component is• a prerequisite for the specific demonstration of antibodies during the early stages of F. Hepatica infection.
The antigenic fractions employed for the iodination were checked for specificity• by immunodiffusion according to Ouchterlony using sheep and bovine sera from animals infected with common gastric and intestinal nematodes, guinea-pig and rabbit sera from animals infected experimentally with Fascioloides magna and bovine sera from animals infected with Cysticercus hovis.Although the results were invariably negative, the F. hepatica antigens prepared in this study cannot be regarded as pure fractions containing only functional antigens.
The quality of the antigen and the protein-bound radioiodine percentage of its protein fraction were considerably affected by the onset of autolytic processes in the flukes.Therefore it is necessary that flukes for the production ov F. hepatica antigen should be isolated from infected animals immediately after slaughter and thorougly washed and frozen without delay.

Fig. 2 .
Fig. 2. Gel filtration on Sephadex G-200 of the crude antigen from adult Fasciola hepatica