Fig 1.
Adaptation of the SA-PolyHRP conjugate and rapid incubations to the MM3-COPRO ELISA.
(A) Two concentrations (5 and 0.62 ng/mL) of whole Fasciola hepatica excretory-secretory antigens (Ag) diluted in CoproGuard were tested with the MM3-COPRO ELISA by using several dilutions of SA-PolyHRP (ranging from 1/10,000 to 1/5,000) as secondary reagent to reveal bound biotinylated mAb MM3. All incubation steps were performed at RT for 30 min with shaking (750 rpm). (B) Two-fold dilutions (0.15–5 ng/mL) of Ag diluted in CoproGuard were assayed with the MM3-COPRO ELISA (SA-PolyHRP diluted 1/8,000) at RT with shaking for several incubation times (30 min, 1 h and 2 h).
Fig 2.
Comparison of three HRP-labeled secondary reagents and several incubation conditions in MM3-COPRO ELISA.
The detectability of the MM3-COPRO ELISA was determined by testing two-fold dilutions (0.15–20 ng/mL) of Fasciola excretory-secretory antigens (Ag) diluted in CoproGuard, and bound mAb MM3 was revealed with (A) NA-HRP, (B) HRP-labeled anti-mouse IgG antibodies (anti-mouse-HRP) or (C) SA-PolyHRP. The following incubation conditions were considered: 37°C (2 h for samples; 90 min and 1 h for secondary reagents; open circles); 4°C (ON) for incubation of samples and 37°C (90 min and 1 h) for subsequent steps (open triangles); and RT (30 min) with shaking at 750 rpm for all incubation steps (closed squares). (D) Combined data obtained with the three secondary reagents and short incubations at RT (30 min, 750 rpm). The colored lines show the cut-off values calculated for anti-mouse-HRP (OD = 0.224; blue line) and SA-PolyHRP (OD = 0.055; red line) by using negative samples from cattle.
Fig 3.
Detectability of rapid MM3-COPRO ELISA performed with three HRP-labeled secondary reagents and two HRP substrates.
Two-fold dilutions (0.15–20 ng/mL) of Fasciola hepatica excretory-secretory antigens (Ag) diluted in fecal supernatants obtained from pooled negative samples from cattle (prepared with distilled water) were tested under short incubations (30 min, 750 rpm) at RT with (A) HRP-labeled anti-mouse IgG antibodies (triangles; cut-off OD = 0.224, blue line), SA-PolyHRP (squares; cut-off OD = 0.055, red line) and NA-HRP (circles), revealed with OPD substrate; and (B) SA-PolyHRP revealed with two HRP substrates, OPD (squares; cut-off OD = 0.055, dashed red line) and TMB (circles; cut-off OD = 0.084, dashed blue line).
Fig 4.
MM3-COPRO OD values obtained for individual Fasciola hepatica positive and negative cattle and sheep feces.
(A) F. hepatica positive (n = 18, closed circles) and negative (n = 30, open circles) samples from cows tested with several variations of the MM3-COPRO test (models A-D). (B) F. hepatica positive (n = 10, closed circles) and negative (n = 20, open circles) samples from sheep tested with models B and C. Model A: classic MM3-COPRO test, performed with standard incubations and a former batch of HRP-labeled anti-mouse IgG antibodies. Model B: performed with short incubations (30 min at RT with shaking), using SA-PolyHRP and OPD. Model C: same as model B, using TMB as substrate. Model D: performed with short incubations and a current batch of HRP-labeled anti-mouse IgG antibodies. The red lines correspond to the cut-off value of each ELISA model: 0.059 for model A (cattle); 0.055 (cattle) and 0.064 (sheep) for model B; 0.084 (cattle) and 0.065 (sheep) for model C; and 0.224 (cattle) for model D.
Fig 5.
Correlation between MM3-COPRO models and fluke burden.
Each point represents an individual cow naturally infected with Fasciola hepatica (n = 18) and tested with several variations of the MM3-COPRO test. (A) Plot of the OD values measured by MM3-COPRO model B (SA-PolyHRP and OPD, rapid incubations) against model A (HRP-labeled anti-mouse IgG antibodies, former batch, and OPD, standard incubations). (B) Plot of the OD values obtained with model C (SA-PolyHRP and TMB, rapid incubations) against model A. (C) Plot of the OD values measured by model C against model B. (D) Plot of the OD values determined by model B against the number of flukes recovered at necropsy.
Fig 6.
Fecal egg counts, fluke burden and OD values obtained by testing fecal samples from cows naturally infected with Fasciola hepatica (n = 18) with the rapid enhanced MM3-COPRO ELISA.
Fluke burdens were determined at necropsy. MM3-COPRO ELISA was performed with short incubations (30 min at RT with shaking) and with SA-PolyHRP as secondary reagent, which was revealed with TMB (model C).
Fig 7.
OD values obtained with the rapid enhanced MM3-COPRO ELISA testing fecal samples from animals experimentally infected with Fasciola hepatica.
(A) Lambs were experimentally infected with 5 (n = 3) or 10 (n = 3) metacercariae. (B) Cows were infected with 25 (n = 2) and 50 (n = 2) metacercariae (Mc). Fecal samples were obtained weekly from each animal during the period of study and analyzed with the MM3-COPRO test using SA-PolyHRP and OPD and short incubations (model B). The cut-off value is 0.064 for sheep and 0.055 for cattle.