Fig 1.
Generation of monoclonal antibodies against rEgP29.
(A) Flow chart for the preparation of anti-rEgP29 positive hybrydoma cell lines. (B) Subtypes of each mAb were determined by ELISA. (C) The antibody titers of each mAb were measured by ELISA. (D) Each mAb recognized the rEgP29 protein. **p < 0.01, ****p < 0.0001.
Fig 2.
Purification and characterization of 4G10F4 mAb.
(A) SDS-PAGE analysis of purified 4G10F4 mAb from hybridoma cell culture supernatant. (B) 4G10F4 mAb reacted with proteins from E. granulosus materials. M: protein marker, Lane PSC: protosoleses; Lane HCW: hydatid cyst wall; HCF: hydatid cyst fluid; Lane rEg P29: recombinant echinococcus granulosis P29. (C) Confocal microscopy images of the hydatid cyst P29 recognized by 4G10F4 mAb. (D) Epitope identification of 4G10F4 mAb by peptide scanning. The core sequence is 61NIASKVADAFQKNKE75. (E) The putative three-dimensional structures of Eg P29 and the anti-P29 antibody were generated, and the binding site between the antigen and antibody was predicted. The black dotted line framed area indicates location of antigen-antibody interaction site.. ****p < 0.0001.
Table 1.
Nucleotide and amino acid sequences of complementary determining regions (CDRs) of 4G10F4 mAb.
Table 2.
Affitiny of P29 antibody to rEg P29 measured by SPR.
Fig 3.
The in vitro effects of 4G10F4 on the viability of E. multilocularis protoscoleces.
(A) The viability of protoscoleces were determined by eosin exclusion. (B) Direct effects of 4G10F4 on the viability of protoscoleces. (C) Effects of 4G10F4 on the viability of protoscoleces in the present of PBMC. PBMC was used as negative control. (D) Effects of 4G10F4 on the viability of protoscoleces in the present of complement. ABZ was used as positive control in protoscoleces viability analysis. ABZ: albendazole; PBMC: peripheral blood mononuclear cell; Com: complement. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no statistical significance.
Fig 4.
4G10F4 showed CDC activity on the E. multilocularis primary cells.
(A) Cell growth was visualised under a light microscope after 24 hours of intervention. (B) Cell viability was assessed via CCK8 assay. (C) Live cells stained with CFSE (green) and dead cells stained with PI (red) were observed using fluorescence microscope. (D) The percentage of the dead cells were calculated using Image J software. Red circled areas indicate cell clones. Em: Echinocooccus multilocularis; ABZ: albendazole; Com: complement. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no statistical significance.
Fig 5.
Evaluation of therapy effects of 4G10F4 on E.multilocularis intraperitoneal infected mice.
(A) Schematic presentation of animal experimental protocol for the 4G10F4 anti-parasitic effects on Em intraperitoneal infected mice (n = 9/group). (B) Gross morphology of metacestode lesions. (C) Quantitative weight analysis of the E.multilocularis metacestode tissues. (D and E) Comparison of spleen and liver index between groups after treatment. Em: Echinococcus multilocularis; PSC: protoscolex; ABZ: albendazole; i.v: intravenous; ig: intragastric gavage; QW: once a week; QD: once a day. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no statistical significance.
Fig 6.
Schematic presentation of animal experimental protocol for the 4G10F4 anti-parasitic effects on infected mice through portal vein (n = 5/group).
Em: Echinococcus multilocularis; PSC: protoscolex; i.v: intravenous; ig: intragastric gavage; QW: once a week; QD: once a day.
Fig 7.
Evaluation of therapy effects of 4G10F4 on E. multilocularis infected mice through portal vein.
(A) Murine liver B-ultrasound images in different groups. The red arrows indicate the metacestode lesions. (B) Gross morphology of metacestode lesions in liver. (C) Comparison of the numbers of infectious foci in the liver surface between groups after treatment. (D and E) Comparison of spleen and liver index between groups after treatment. (F) Histopathological changes were observe by H&E staining and PAS staining. H&E: hematoxyli and eosin; PAS: Periodic Acid-Schiff; GL: germinal layer; LL: laminated layer; PSC: protoscolex; PV: parasitic vesicle. *p < 0.05, **p < 0.05, ns: no statistical significance.