A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst

Abstract

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R2 = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R2 = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.

A comparison of Plasmodium falciparum circumsporozoite protein-based slot blot and ELISA immuno-assays for oocyst detection in mosquito homogenates

“… Preparation of whole mosquito homogenates : CSP expression on the developing oocysts was determined in lysed whole mosquitoes using a procedure described previously [ 27 ]. Briefly, individual blood-fed or unfed mosquitoes were placed into single tubes and homogenized with a piston in 50 μl of lysis buffer (1× TBS, 0.5 % SDS).…”

“…If the ELISA is performed at 7 days PI, when oocysts are visible by microscopy, CSP expression by developing oocysts is too low for detection [ 11 ]. The enhanced chemi-luminescent ELISA (ECL ELISA) and slot-blot assays (ECL-SB) were designed to overcome these issues of sensitivity, and are capable of detecting as little as 4.4 and 1 pg of recombinant CSP, respectively [ 27 , 28 ], which could allow for Plasmodium detection shortly after the onset of CSP production during oocyst development.…”

“…Gametocyte cultures were diluted to ensure parasite burden in test mosquitoes was close to the level of Plasmodium -infected vectors from endemic regions [ 29 , 30 ], providing groups of mosquitoes with controlled, but varying infection prevalence to investigate the performance of the two assays relative to microscopy for the detection of oocyst-stage infections. Since CSP expression increases during oocyst maturation [ 27 , 31 ], the sensitivity of the assays in longer term infected mosquito specimens that contain a higher amount of CSP was also examined.…”

A synthetic promoter for multi-stage expression to probe complementary functions of Plasmodium adhesins

“…In addition, we observed early oocysts that had not yet initiated sporogony, which showed no mCherry. This indicates a shift to later expression compared to that of CSP, which is already expressed in early oocysts (Simonetti et al, 1993;Kumar et al, 2014;Stone et al, 2015). The amount of mCherry in liver stages was low and only detectable 24 h but not 48 h after infection ( Fig.…”

Transmission blocking malaria vaccines: Assays and candidates in clinical development

“…Two outcome measures of the SMFA are the proportion of infected mosquitoes (oocyst prevalence) and the infection burden (oocyst density) in mosquitoes. These outcomes are commonly determined by microscopical examination of mosquito midgut but can also be determined by the detection of parasite DNA by molecular methods [64,65] or parasite protein by immuno-assays [65,66]. Recently, fluorescence and luminescence-based SMFA approaches have been proposed that use P. falciparum strains expressing green fluorescent protein [67] and firefly luciferase protein [68].…”