Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens

Abstract

ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1–10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis.

Fasciola and fasciolosis in ruminants in Europe: Identifying research needs

“…(, ) showed that this test has a high diagnostic specificity (100%), and showed no cross reaction when tested with soluble fractions of homogenates from Paramphistomum cervi and Taenia hydatigena but also in co‐infections with paramphistome, coccidian and/or gastrointestinal nematodes. However, the sensitivity of this ELISA can sometimes be compromised by the high variability in the concentration of cathepsin proteinases in faecal samples and by differences in the between‐batch performance of peroxidase‐labelled anti‐mouse IgG polyclonal antibodies (Martínez‐Sernández, Orbegozo‐Medina, González‐Warleta, Mezo, & Ubeira, ). Brockwell, Spithill, Anderson, Grillo, and Sangster () and Palmer, Lyon, Palmer, and Forshaw () improved the sensitivity of MM3‐COPRO ELISA using a customized cut‐off for sheep and cattle whilst maintaining the specificity above 99%.…”

“…However, the test had poor diagnostic sensitivity in horses (Palmer et al., ). Recently a new version of the coproantigen test using a streptavidin‐polymerized horseradish peroxidase conjugate was evaluated and was sufficiently sensitive to detect infection with a single fluke (Martínez‐Sernández et al., ).…”

Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis

“…Two methods were used to calculate the cut-off values for the different indirect ELISAs. In method A, designed to guarantee maximum specificity (i.e., 100%; see Martínez-Sernández et al 2016), the cut-off values were calculated as the sum of the highest OD value obtained on testing the negative sera plus 1SD. These cut-offs were used to obtain the sensitivities of each test, which were calculated using EPIDAT 3.1 software as the number of true positives (correctly identified by each test), divided by the total number of infected animals.…”

“…In addition to fluke counts, samples of feces from all animals were subjected to sedimentation (Anderson et al 1999) and flotation (MAFF 1971) procedures to concentrate eggs from Fasciola or other parasites, and then they were microscopically examined. The remaining fecal material was frozen at − 25°C for subsequent further quantitation of Fasciola antigens by the MM3-COPRO ELISA (Mezo et al 2004;Martínez-Sernández et al 2016).…”

“…As for cattle samples, the feces from all sheep were examined for the presence of eggs from Fasciola and other parasites. Fecal aliquots were also frozen at − 25°C for further quantitation of Fasciola antigen by the MM3-COPRO ELISA (Mezo et al 2004;Martínez-Sernández et al 2016).…”