title: Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols.
abstract: Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.
authors.affiliation: 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba .
authors.forename: Annia
authors.initials: A
authors.lastname: Alba
authors.affiliation: 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba .
authors.forename: Ricardo
authors.initials: R
authors.lastname: Marcet
authors.affiliation: 2 Laboratorio de Anticuerpos Monoclonales, Vicepresidencia de Investigaciones , Instituto Finlay, La Habana, Cuba .
authors.forename: Oscar
authors.initials: O
authors.lastname: Otero
authors.affiliation: 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba .
authors.forename: Hilda M
authors.initials: HM
authors.lastname: Hernández
authors.affiliation: 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba .
authors.forename: Mabel
authors.initials: M
authors.lastname: Figueredo
authors.affiliation: 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba .
authors.forename: Jorge
authors.initials: J
authors.lastname: Sarracent
mesh_terms: D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D001201:Ascites / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005210:Fasciola hepatica / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology; D008807:Mice, Inbred BALB C
chemical_list: D000909:Antibodies, Helminth; D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M