2 research outputs found
Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle
Background A major constraint for the effective control and management of
helminth parasites is the lack of rapid, high-throughput, routine diagnostic
tests to assess the health status of individual animals and herds and to
identify the parasite species responsible for these helminthoses. The
capability of a multiplex platform for the simultaneous detection of three
pasture associated parasite species was evaluated and compared to existing
ELISAs. Methods The recombinant antigens 14.2 kDa ES protein for Cooperia
oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for
Fasciola hepatica were recombinantly expressed either in Escherichia coli or
Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal
concentrations for coupling were determined following the examination of serum
samples collected from experimentally mono-infected animals, before and after
their infection with the target species. Absence of cross-reactivity was
further determined with sera from calves mono-infected with Haemonchus
contortus, Ostertagia ostertagi and Trichostrongylus colubriformis.
Examination of negative serum samples was characterised by low median
fluorescence intensity (MFI). Results Establishment of the optimal serum
dilution of 1:200 was achieved for all three bead sets. Receiver Operating
Characteristic analyses were performed to obtain cut-off MFI values for each
parasite separately. Sensitivity and specificity at the chosen cut-off values
were close to, or 100 % for all bead sets. Examination of serum samples
collected on different days post infection from different animals showed a
high reproducibility of the assays. Serum samples were additionally examined
with two already established ELISAs, an in-house ELISA using the recombinant
MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The
results between the assays were compared and kappa tests revealed an overall
good agreement. Conclusions A versatile bead-based assay using fluorescence
detection (xMAP® technology) was developed to simultaneously detect antibodies
against C. oncophora, D. viviparus and F. hepatica in cattle serum samples.
This platform provides rapid, high-throughput results and is highly sensitive
and specific in comparison to existing serological as well as coproscopical
diagnostic techniques