Fig. 4. (A) High-performance liquid chromatography (HPLC) fractionation of an acetic acid extract from 50 SGequiv of Astacus leptodactylus. A reverse-phase column (Nucleosil C-18; 250 mm length x 4.6 mm i.d.; 5 µm particle size) was used. Eluant A: 0.1% trifluoroacetic acid (TFA) in water; eluant B: 0.1% TFA in 100% acetonitrile. The elution gradient of eluant B is indicated by the dotted line. Flow rate was 750 µl min^-1 and fraction duration was 1 min. UV detection was set at 220 nm. Arrows P1, P2 and P3 show the major immunoreactive peaks. Fractions 45-48 were pooled in zones Z1 (fractions 45+46), Z2 (fraction 47) and Z3 (fraction 48) for bioassay. (B—D) Results of the enzyme-linked immunosorbent assay (ELISA) tests performed on 5 µl of each HPLC fraction with different antisera: (B) anti-Astacus crustacean hyperglycemic hormone (CHH); (C) anti-D antiserum; (D) anti-L antiserum. The columns represent the absorbance determined at 405 nm after 120 min.