Molecular characterization of cloned SmANX 18. (a) Determination of the optimal antigen coating concentration. (b) Determination of anti-rSmANX18 immune serum titer by indirect ELISA. Red, green, blue, and light blue represent serum dilutions of 1:10³, 1:10⁴, 1:10⁵, and 1:10⁶, respectively. (c) SDS-PAGE analysis of 10 μg purified ANX from S. mansoni on a 10% gel. M: protein prestaining marker; Lane 1: uninduced bacterial cultures; Lane 2: the lysate of the induced recombinant bacteria harboring PMAL-c2X-rSmANX 18 after ultrasonication; Lane 3: rSmANX 18 purified by amylose resin column. (d) rSmANX 18 antigenicity analysis. M: protein prestaining marker; Lane 1: rSmANX 18 + anti-rSmANX 18 serum; Lane 2: rSmANX 18 + infected mouse serum; Lane 3: rSmANX 18 + preimmune serum. (e) The transcription pattern of the ANX gene in different developmental stages of Spirometra mansoni by conventional RT-PCR. The cDNA from various developmental stages of S. mansoni, including eggs, plerocercoid and immature proglottids, mature proglottids, and gravid proglottids of adult worms. A housekeeping gene (Sm-GAPDH) was used as a positive control. H₂O was used as a negative control. (f) rSmANX 18 expression of S. mansoni in different stages by qRT-PCR. Blue represents plerocercoid; red represents IMP; purple represents MP; green represents GP; orange represents egg. IMP, immature proglottid; MP, mature proglottid; GP, gravid proglottid. The expression level was normalized to GAPDH and measured with the 2^−ΔΔCt method. The results were averaged from three independent replicates during all stages. Error bars represent SD (n = 3).