A slot blot immunoassay for quantitative detection of Plasmodium falciparum circumsporozoite protein in mosquito midgut oocyst.

Sanjai Kumar,Kazutoyo Miura,Bryan Grabias,Ashley Birkett,Babita Mahajan,Emily Locke,Yukiko Kozakai,Merribeth J Morin,Hong Zheng,Bingbing Deng,Carole Long,Clive Shiff

doi:10.1371/journal.pone.0115807

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.

Highlights

Sensitive and reproducible immunoassays that are amenable to high throughput adaptation are a critical need for diagnostics, vaccine development and manufacturing, and in pathogen surveillance and epidemiology studies
Recombinant P. falciparum circumsporozoite protein (CSP) amino acid sequence 27123[NANPNVDP]3[NANP]21300-411 expressed in E. coli and purified on a heparin sepharose affinity column was used as source of recombinant antigen
In order to facilitate the development of CSP-based immunoassays for parasite detection in the midgut, we wanted to know the dynamics of CSP expression during the midgut developmental stages

Summary

Introduction

Sensitive and reproducible immunoassays that are amenable to high throughput adaptation are a critical need for diagnostics, vaccine development and manufacturing, and in pathogen surveillance and epidemiology studies. ECL Slot Blot Assay for Plasmodium Detection populations that distinctly contained either high and low midgut Pf Oocyst burden. One report has utilized a transgenic luciferase-expressing P. falciparum strain for high throughput measurement of oocyst burden in blood meal fed mosquitoes in the laboratory setting [10].

Results

Conclusion