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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 (r Pf CSP) and native  Pf CSP from Oocysts ( Pf  Oocyst) developing in the midguts of  Anopheles stephensi  mosquitoes. The ECL-SB detects as little as 1.25 pg of r Pf CSP (linear range of quantitation 2.5–20 pg; R 2  = 0.9505). We also find the earliest detectable expression of native  Pf CSP 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  Oocyst s  (linear quantitation range 1–4, R 2  = 0.9795) and determined that one  Pf  Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native  Pf CSP, 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 r Pf  CSP and native  Pf CSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for r Pf  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 Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst