Physiological, morphological, and immunochemical parameters used for the characterization of clinical and environmental isolates ofAcanthamoeba

Abstract

The factors that characterize Acanthamoeba strains as harmless or potentially pathogenic have not been elucidated. Analysing the in vitro and in vivo parameters of Acanthamoeba samples, including heat tolerance at temperatures close to that of the human body, cytopathic effects, and their ability to cause infections in animals, has been proposed to identify their pathogenic potential. Another promising criterion for differentiating strains is the analysis of their biochemical and immunochemical properties. In this study, a comparative evaluation between clinical and environmental Acanthamoeba isolates was performed on the basis of physiological, morphological, and immunochemical criteria. Crude antigens were used to characterize the protein profiles by electrophoresis and immunize mice to produce polyclonal and monoclonal antibodies. The antibodies were characterized using ELISA, Western blotting, and immunofluorescence techniques. The results obtained with polyclonal antibodies suggest the presence of specific proteins for each studied isolate and co-reactive immunochemical profiles among conserved components. Ten monoclonal antibody clones were obtained; mAb3 recognized 3 out of 4 samples studied. The results of this study may help standardize criteria for identifying and characterizing Acanthamoeba strains. Taken together, our results support the view that a set of features may help differentiate Acanthamoeba species and isolates.

Acanthamoeba spp. monoclonal antibody against a CPA2 transporter: a promising molecular tool for acanthamoebiasis diagnosis and encystment study

“…Antibody-secreting hybridomas specific against Acanthamoeba antigens were produced by Becker-Finco et al . (2013), including mAb3. The cells of interest were defrosted and remained secreting reactive antibodies when cultivated in optimal conditions.…”

“…The confirmation that the mAb3 target is on the cell surface supports the idea of employing it in immunofluorescence tests for detection of Acanthamoeba trophozoites. Some laboratories are already using immunofluorescence to aid the diagnosis of AK (Lorenzo-Morales et al ., 2015) and several studies utilize this technique to detect trophozoites (Khan et al ., 2000; Magnet et al ., 2012; Becker-Finco et al ., 2013; Kang et al ., 2018). However, most of them use polyclonal antibodies and all apply the indirect detection method, which increases the chances of non-specific reactivity and make the protocol more laborious.…”

“…However, the identification of new relevant targets in the context of Acanthamoeba physiology is also necessary for a better comprehension of pathogenesis, which is yet not completely understood (Neelam and Niederkorn, 2017;Kalra et al, 2020). Becker-Finco et al (2013) previously produced the monoclonal antibody mAb3, which can recognize only pathogenic Acanthamoeba isolates by enzyme-linked immunosorbent assay (ELISA), Western Blotting (WB) and indirect immunofluorescence. The present study aimed to evaluate mAb3's capacity of directly detecting Acanthamoeba trophozoites by flow cytometry and immunofluorescence conjugating it to a fluorophore.…”

“…Becker-Finco et al . (2013) previously produced the monoclonal antibody mAb3, which can recognize only pathogenic Acanthamoeba isolates by enzyme-linked immunosorbent assay (ELISA), Western Blotting (WB) and indirect immunofluorescence. The present study aimed to evaluate mAb3's capacity of directly detecting Acanthamoeba trophozoites by flow cytometry and immunofluorescence conjugating it to a fluorophore.…”

Generation and characterization of monoclonal antibody against Advanced Glycation End Products in chronic kidney disease

“…IGH-For (GAC AGT GGA TAR ACM GAT GG) and IGH-Rev (GAG GTS MAR CTG CAG SAG TCW GG) for mAb 2D6G2 VH amplification, and V-KAPPA For (GGA TAC AGT TGG TGC AGC ATC) and V-KAPPA Rev (GAT ATT GTG CTA ACT CAG TCT) for mAb 2D6G2 light chain variable VL amplification. PCR products were inserted into the cloning vector pGEM-T (Promega A3610) before being sequenced and analyzed according to the protocol previously reported [17] .…”