Isolamento dal siero di maiale di globuline con attività del terzo componente del complemento e caratterizzazione immunochimica di esse

Tolone, Giuseppe ; Ciccimarra, Filippo ; Pontieri, Giuseppe Mario

Atti della Accademia nazionale dei Lincei. Rendiconti della Classe di scienze fisiche, matematiche e naturali, Série 8, Tome 41 (1966) no. 6, pp. 565-573

Résumé

Following addition of "Liquoid" (Sodium polyanetholsulphonate) to serum of different animal species, a precipitate is formed resulting of complexes between serum basic proteins and "Liquoid" itself. In the precipitate are enclosed globulins furnished with C'3 activity so that the procedure is routinely employed for the preparation of the so called R3, i.e., serum deprived of C'3 activity which can be successfully employed for the titration of such a component of complement in a given serum. The present investigation was undertaken in order to show whether or not it is possible to obtain from the above mentioned complexes isolation and purification of the third component of complement and, if such were the case, to study also the possibility of immunization of animals of different species for the production of antibodies specifically reacting with C'3. Pig serum was selected as source of C' because of its richness in the third component. A method is described which leads to the isolation of C'3 activity from the complexes obtained by addition of "Liquoid" to pig serum. The procedure is based on the assumption that the affinity of "Liquoid" for basic proteins, as lysozyme or protamine sulphate, is higher than that against serum proteins. Following suspension of the precipitate resulting from serum globulins- "Liquoid" complexes in a medium containing either lysozyme or protamine sulphate, globulins, furnished with C'3 activity, become again soluble, free from the complexes with "Liquoid". After separation of the precipitate, the supernatant obtained by such a procedure was filtered through Sephadex G-100. Among the several fractions obtained one was shown to contain C'3 activity, without presence of other components of complement, as demonstrated by its ability to lyse sensitized sheep erythrocytes (EA) in the presence of only a R3 reagent or to lyse EAC'1,4,2 in the presence of EDTA. Further investigations demonstrated the possibility of obtaining in rabbits antibodies specifically reacting with pig serum globulins furnished with C'3 activity. By addition of the immune serum to either pig serum or to the fraction isolated by filtration through Sephadex G-100, inhibition of the third component of complement takes place. The precipiting reaction of these antibodies with the corresponding antigens was studied by help of immunodiffusion and immunoelectrophoretic methods.