Fig 1.
Determination of the specificity of MoAb 4E3 with CE from F. gigantica, other trematode, cestode and nematode parasites by using indirect ELISA and immunoblot analysis.
(A) ELISA OD values of cross-reactivities of MoAb 4E3 and antigens from various trematode, cestode and nematode species. Fg = F. gigantica, Fh = F. hepatica, Ge = G. explanatum, Ep = E. pancreaticum, Pc = P. cervi, Pg = P. gracile, Cc = C. cotylophorum, Fc = F. cobboldi, Gc = G. crumenifer, Ss = S. spindale, Mb = M. benedeni, Ac = A. centripunctata, Ts = Trichuris sp., Hp = H. placei and Sp = S. labiato-papillosa, Sm = S. mansoni, Sme = S. mekongi, Sj = S. japonicum, and Ov = O. viverrini. Undil = undiluted MoAb hybridoma fluid, MoAb dilution = MoAb hybridoma fluid diluted at 1:10, 1: 100, 1: 500 and 1: 1,000. The cut-off value is indicated by a horizontal dashed line and calculated as the mean OD of negative controls plus 3 SD. The OD values greater than this cut-off value are considered to be positive. (B) Immunoblotting detection of the cross-reactivities of MoAb 4E3 with CE from F. gigantica and other trematode parasites. Fg = F. gigantica (lane 2), Fh = F. hepatica (lane 3), Ge = G. explanatum (lane 4), Ep = E. pancreaticum (lane 5), Pc = P. cervi (lane 6), Pg = P. gracile (lane 7), Cc = C. cotylophorum (lane 8), Fc = F. cobboldi (lane 9) and Gc = G. crumenifer (lane 10) and Ss = S. spindale (lane 11), (C) Immunoblotting detection of the cross-reactivities of MoAb 4E3 with CE from F. gigantica, other trematode, cestode and nematode parasites. Mb = M. benedeni (lane 3), Ac = A. centripunctata (lane 4), Ts = Trichuris sp. (lane 5), Hp = H. placei (lane 6), Sp = S. labiato-papillosa (lane 7), Sm = S. mansoni (lane 8), Sme = S. mekongi (lane 9), Sj = S. japonicum (lane 10) and Ov = O. viverrini (lane 11). Lane 1 of B and C is CE from F. gigantica blotted with the myeloma culture fluid (MCF), which is used as the negative control. STD is the lane containing standard protein molecular weight markers indicated on the left side.
Fig 2.
Immunofluorescence staining of CatL1 proteases of adult F. gigantica using the specific MoAb 4E3 as a probe.
(A) The negative control of a cross section stained with myeloma culture fluid, showing tegument (Te), spine (Sp), muscle (Mu), caecum (Ca), and parenchymal cells (Pc), while vitelline glands (Vi) appear only nonspecific orange autofluorescence. (B) A low magnification micrograph showing intense fluorescence in both caecal epithelium and in the lumen of the caecum, while the tegument (Te), spine (Sp), muscle (Mu) and parenchymal cells (Pc) are not stained. (C and D) Medium and high magnification micrographs showing intense fluorescence in both caecal epithelium and in the lumen of the caecum, while parenchymal cells (Pc) are not stained.
Fig 3.
A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) is developed for diagnosis of fasciolosis by F. gigantica.
(A and B) The lowest concentrations of rCatL1, CatL1 in CE of variuos stages of F. gigantica, and in ES antigens of adult F. gigantica as detected by the sandwich ELISA. (A) Lines with black circle, white triangle and black square denote the concentration levels of rCatL1 and CatL1 in CE and ES antigens of adult F. gigantica. The arrows indicate the lowest concentrations of CatL1 that could still be detected. (B) Lines with black circle, white circle, black triangle, white triangle, black square and white square show the concentration levels of CatL1 in CE of adult, 5-, 3-, 1-week-old juveniles, NEJ and Met of F. gigantica, respectively. The arrows indicate the lowest concentrations that CatL1 could still be detected. (C) Detection of circulating CatL1 antigens in the serum samples of mice experimentally infected with F. gigantica as compared with non-infected mice by sandwich ELISA. Black circles denote OD values of individual mouse serum; white squares represent mean OD values of all mice in each experimental group. The horizontal dotted line represents the cut-off value for a positive detection. (D) The relative levels of circulating CatL1 antigens in the serum samples from mice infected with F. gigantica and S. mansoni as well as hamster infected with O. viverrini as examined by sandwich ELISA (OD values at 450 nm). The serum samples from non-infected mice and hamsters are used as negative controls. The horizontal dotted line is the cut-off value for a positive detection. (E) The relative levels of circulating CatL1 antigens in the serum samples from cattle naturally infected with F. gigantica (fasciolosis), P. cervi (paramphistomosis), M. benedeni (Monieziasis), Strongylids (strongylid infection), Trichuris sp. (Trichuriasis), and Strongyloides sp.(strongyloidiasis) as measured by sandwich ELISA (OD values at 450 nm). Serum samples from non-infected cattle are used as negative controls. The horizontal dotted line represents the cut-off value for a positive detection.
Table 1.
Specificity testing of a sandwich ELISA to various crude preparations from trematode, cestode, and nematode parasite antigens.
Fig 4.
An immunochromatographic (IC) strip test is developed for diagnosis of fasciolosis by F. gigantica: Experiment trial.
(A) A schematic diagram of the immunochromatographic (IC) strip test showing several components: a sample pad, a conjugate pad, an immobilized nitrocellulose membrane (control and test antibody) and an absorbent pad. (B) The samples of the IC strip test for deciding the results: a positive result shows two red dots at the test and control regions, while a negative result exhibits only one red dot in the control region. The strip tests are invalid when there is no red dot at the control region. (C-E) Sensitivity testing of the IC strip was studied using a series of dilutions in a buffer (0.0512–20,000 ng/ml) of adult F. gigantica recombinant cathepsin L1 (rCatL1) (C), crude extract (CE) (D), and excretory-secretory (ES) antigens (E). 1 = 20,000 ng/ml, 2 = 4,000 ng/ml, 3 = 800 ng/ml, 4 = 160 ng/ml, 5 = 32 ng/ml, 6 = 6.4 ng/ml, 7 = 1.2 ng/ml, 8 = 0.256 ng/ml, 9 = 0.0512 ng/ml, and 10 = buffer control. (F) The specificity of the IC strips were tested against CE from F. gigantica, other trematode, cestode and nematode parasites. All antigens were tested at 1,000 μg/ml. Fg = F. gigantica, Bf = buffer control, Fh = F. hepatica, Ge = G. explanatum, Ep = E. pancreaticum, Pc = P. cervi, Pg = P. gracile, Cc = C. cotylophorum, Fc = F. cobboldi, Gc = G. crumenifer, Ss = S. spindale, Mb = M. benedeni, Ac = A. centripunctata, Ts = Trichuris sp., Hp = H. placei, Sp = S. labiato-papillosa, Sm = S. mansoni, Sme = S. mekongi, Sj = S. japonicum and Ov = O. viverrini.
Fig 5.
A field trial of the immunochromatographic (IC) strip test for diagnosis of fasciolosis by F. gigantica.
(A) Detection of circulating CatL1 antigens in the serum samples of mice experimentally infected with F. gigantica as compared with non-infected mice. D1 = day 1 post infection, D4 = day 4 post infection, D7 = day 7 post infection, D = day 21 post infection, D35 = day 35 post infection, IS = infected serum, and NS = normal serum. (B) Detection of circulating CatL1 antigens in the serum samples of cattle naturally infected with F. gigantica (strip 1), P. cervi (strip 2), M. benedeni (strip 3), Strongylids (strip 4), Trichuris sp. (strip 5), and Strongyloides sp. (strip 6). Serum sample from non-infected cattle was used as the negative control (strip 7). (C) Detection of circulating CatL1 antigens in the serum samples of mice experimentally infected with F. gigantica as compared with non-infected mice, showing results of positivity (+VE) and negativity (-VE) as measured by IC test. (D and E) Comparison between positivity (+VE) and negativity (-VE) of different parasite species as measured by IC test. (D) Sera from mice experimentally infected with fasciolosis (1), opisthorchiasis (2), schistosomiasis (3), non-infected mice (4), and non-infected hamsters (5). (E) Sera from cattle naturally infected with fasciolosis (1), paramphistomosis (2), monieziasis (3), strongylid infection (4), trichuriasis (5), strongyloidiasis (6) and non-infected cattle (7).
Table 2.
A comparison of diagnostic values of the sandwich ELISA and IC test for CatL1 antigen detection in sera of mice experimentally and cattle naturally infected with F. gigantica.