RT Journal Article
T1 Rapid enhanced MM3-COPRO ELISA for detection of fasciola coproantigens
A1 Martínez Sernández, Victoria
A1 Orbegozo Medina, Ricardo Alfredo
A1 González Warleta, Marta
A1 Mezo, Mercedes
A1 Martínez Ubeira, Florencio César
K1 MM3-COPRO ELISA
K1 Fasciola Coproantigens
AB ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniquesfor diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-houseMM3-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 andfor monitoring the efficacy of anthelmintic treatments in human and animal species, as theyprovide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentrationof 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-mouseIgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in orderto reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of theMM3-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 ODvalues can be achieved with short incubations (30 min each step) at RT with shaking, ratherthan standard incubations, which significantly accelerates the diagnostic procedure. Finally,we did not find a significant correlation between coproantigen concentration and parasiteburden 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 fordetecting animal and human infections has already been demonstrated, it is expected thatthe improvements reported in this study will add new insights into the diagnosis and controlof fasciolosis
AB We have previously reported how the combined use of mAb MM3 with polyclonal antibodies obtained from rabbit immunized with Fasciola hepatica excretory-antigens led to the development of the in-house MM3-COPRO ELISA and its commercialversion BIO K 201 (BIO X Diagnostics, Belgium), which are widely used to detect humanand animal infections caused by F. hepatica. After more than a decade in use, both testshave proven to be useful tools for specifically detecting Fasciola infections, although it hasalso been found that: i) the conditions of use of the commercial test in some field studiesdid not enable the sensitivity obtained with the in-house test to be reached, and ii) thebatches of the secondary reagent (peroxidase-labeled anti-mouse antibodies) currentlyavailable for use in the in-house test do not perform the same as previous batches. Tosolve these problems, we provide data showing that the incorporation of an enhancementsystem consisting of streptavidin-polymerized horseradish peroxidase conjugate greatlyimproved the sensitivity of the MM3-COPRO ELISA and enabled reduction of the incubation time. These modifications enabled the detectability of the assay to be 150 pg/mL, thusenabling detection of infection in animals harboring only one fluke
PB Plos
SN 1935-2727
YR 2016
FD 2016
LK http://hdl.handle.net/10347/22985
UL http://hdl.handle.net/10347/22985
LA eng
NO Martínez Sernandez, V., Orbegozo Medina, R.A., González Warleta, M., Mezo, M. and Ubeira, F.M. (2016). Rapid enhanced MM3-COPRO ELISA for detection of fasciola coproantigens. Plos Neglect. Trop. Dis.,  vol. 10 (7): e0004872
NO This work was funded by Ministerio deCiencia e Innovación, Spain, Grants AGL2011-30563-C02/C03; Xunta de Galicia, Spain, GrantGPC2014/058; and the European Fund for RegionalDevelopment (FEDER). VMS holds a predoctoralfellowship from the Spanish Ministerio de Educación,Cultura y Deporte (Programa de Formación delProfesorado Universitario, AP2010-5808) and RAOMis recipient of a fellowship from the Spanish Ministeriode Economía y Competitividad (Programa deFormación de Personal Investigador, BES-2012-060270)
DS Minerva
RD 24 abr 2026