Plasmodium vivax: detection of blood parasites using fluorochrome labelled monoclonal antibodies

Abstract

A monoclonal antibody (MoAb) 2C6111 specific for Plasmodium vivax erythrocytic stages was shown to detect parasitized erythrocytes in blood samples collected in the field. This MoAb binds to the mature trophozoite, schizont and gametes of P. vivax and upon examination of 43 wild isolates no evidence of polymorphism was found. To search for P. vivax parasites in human blood a MoAb immunofluorescent test (MoAb-IFT) was developed. The assay is based on the ability of fluorescein isothiocyanate labelled MoAb 2C6111 to combine with parasitized erythrocytes on thin blood smears. A preliminary field trial was carried out in Venezuela to determine the usefulness of MoAb-IFT for the specific diagnosis of P. vivax malaria. Blood samples collected from malarious and non-malarious individuals were examined both by standard microscopy of Giemsa stained thick blood smears (G-TS) and MoAb-IFT. The latter was specific and gave a 100% correlation with G-TS. Sensitivity was close to that usually achieved with Giemsa stained blood films.

Caveolins and flotillin-2 are present in the blood stages of Plasmodium vivax

“…Immunofluorescence patterns produced by anti-caveolin-1-3 antibodies on infected erythrocytes were studied using acetone-or 4% paraformaldehyde-0.1% glutaraldehyde (PAF-G) fixed parasites, as previously described (Perez et al 1995). For single-label immunofluorescence, a dilution of the primary antibody was applied to the cell monolayer and incubated for 90 min at room temperature (RT).…”

“…Human erythrocytes infected with P. vivax were obtained from patients with parasitaemias between 0.3 and 1%, as described elsewhere (Perez et al 1995). In brief, after consent was obtained voluntarily from the patients, up to 10 ml of blood was drawn into sterile heparinized tubes.…”

Plasmodium vivax and P. chabaudi: inter-species cross-reacting antigens

“…MAbs were produced in our laboratory by immunization of BALB/c mice with human erythrocytes infected with Plasmodium vivax as described elsewhere (Perez et al 1995). In brief, following the obtainment of voluntary, informed consent, up to 15 ml of blood from a patient infected with P. vivax was drawn into sterile heparinized tubes and then passed through a CF11 cellulose column to remove leukocytes (Richards and Williams 1973).…”

“…Culture supernatants were screened for reactivity with blood stages of P. vivax by indirect immunofluorescence assays (IFA) on acetone-fixed parasites (Perez et al 1995). Positive colonies were cloned twice by limiting dilution and cryopreserved in liquid nitrogen.…”

“…IFAs on parasitized erythrocytes were performed as previously described (Perez et al 1995). In brief, a suspension of parasitized erythrocytes was fixed with a solution containing 4% (w/v) paraformaldehyde (Sigma) and 0.1% glutaraldehyde (EM grade, Polysciences, USA; (PAF/G) in PBS for 10 min at room temperature.…”