A sensitive enhanced chemiluminescent-ELISA for the detection of Plasmodium falciparum circumsporozoite antigen in midguts of Anopheles stephensi mosquitoes

Highlights

• ECL-ELISA is a sensitive method for biomarker detection or pathogen screening in epidemiological studies.

• ECL-ELISA can detect 4.4 pg of recombinant Plasmodium falciparum (Pf) circumsporozoite protein or 5 Pf sporozoites.

• Approximately 0.056 of Pf oocyst can be detected from whole mosquito sample by ECL-ELISA.

• Overall, the ECL-ELISA is a sensitive screening platform which is amenable to high throughput analyses.

Abstract

Efforts to develop a successful malaria vaccine are hampered due to lack of assays that are predictive of protective immunity without conducting large clinical studies. The effect of experimental vaccines and drugs on malaria transmission is yet more difficult to measure. Knowledge on the Plasmodium infection rate in mosquito populations will aid the measurement of effects from intervention measures for malaria control. Here, we report the development of a chemiluminescent sandwich ELISA (ECL-ELISA) that can detect Plasmodium falciparum circumsporozoite protein (Pf CSP) produced in recombinant form at concentrations of 4.4 pg and in P. falciparum sporozoites (Pf SPZ) derived from mosquito salivary glands at levels corresponding to 5 Pf SPZ. Most importantly, we demonstrate reliable Pf CSP-based detection of 0.056 day 8 P. falciparum oocysts developing inside mosquito midguts in whole mosquito lysates. Cumulatively, the ECL-ELISA is 47 × more sensitive for the detection of Pf CSP than a colorimetric ELISA while greatly simplifying sample preparation, obviating the need for cumbersome midgut dissections and allowing high throughput screening of Plasmodium infection in mosquito populations. The ECL-ELISA may also have broader application in diagnosis of infectious diseases and the prognostic value in cancer and other diseases such as auto-immunity and genetic disorders based on antigen detection, or quality validation of biological vaccine components.

Introduction

Malaria is a persistent threat to global public health with an estimated 219 million cases and 627,000 deaths annually (WHO, 2012). Successful propagation of the causative Plasmodium parasites requires a developmental cycle in Anopheles spp. mosquito vectors before being transmitted to the human host through an infectious bite. This critical stage of the parasite life cycle represents a bottleneck or vulnerability in the growth and survival of Plasmodium (Wang and Jacobs-Lorena, 2013) and intervention strategies that specifically target the mosquito host to disrupt the parasite transmission are being designed (Fang et al., 2011, Parvez and Al-Wahaibi, 2003, Rajakumar and Abdul Rahuman, 2011). Such strategies include the novel drugs and vaccines that target the sexual stage of life cycle and application of insecticide-treated nets, residual sprays inside residence and disruption of mosquito habitats. However, the success of any of these mosquito-borne transmission blocking strategies would depend on the availability of effective methods to measure the effect of intervention measures on parasite infection rate in mosquito populations in endemic areas.

Outside of traditional microscopy, the enzyme-linked immunosorbent assay (ELISA) is a commonly used method to screen biological samples for the presence of infectious agents. Indeed, ELISAs are routinely employed to screen for pathogens by the detection of specific antigens and measure antibody responses in patients (Cao et al., 2014, Li et al., 2014), monitor physiological responses to drug treatments (Pereira et al., 2014) and screen for chemical contaminants in the food supply (Quan et al., 2011). The overall sensitivity of these assays is typically limited by the affinity of the antigen–antibody interaction as well as the nature of the reporter system used to quantify the analyte under investigation. A number of studies have established the effectiveness of diagnostic calorimetric ELISA assays in the detection of specific Plasmodium antigens in blood, such as histidine rich protein 2 and lactate dehydrogenase, at levels corresponding to parasite densities of approximately 1–20 parasites/μL or approximately 0.08-2 ng protein/μL (Atchade et al., 2013, Bashir et al., 2013, Martin et al., 2009), and with diagnostic sensitivities and specificities in the 90%–100% range (Atchade et al., 2013, Noedl et al., 2006). Similarly, a few studies have reported on the Plasmodium falciparum circumsporozoite (Pf CSP)-based ELISA for detection of Plasmodium parasites in mosquito vectors (Burkot et al., 1984, Collins et al., 2004). More specifically, standard calorimetric ELISA experiments directed against Pf CSP utilizing whole mosquito lysates or dissected salivary glands have demonstrated detection limits of 3–50 sporozoites/μL (Fontenille et al., 2001). Ongoing optimization of the calorimetric ELISA protocol has further increased the sensitivity of such assays to 0.25 sporozoites/μL, specifically when using purified sporozoites that have been isolated from dissected mosquito salivary glands (De Arruda et al., 2004). In the absence of proper dissection of mosquito glands and midguts before ELISA analysis, a high diagnostic sensitivity of 100% (Fontenille et al., 2001) can be reduced to 71.6% (Beier et al., 1987).

In this study, we report the development of an enhanced chemiluminescent ELISA (ECL-ELISA), which is capable of detecting as few as 5 P. falciparum sporozoites (Pf SPZ) and identifying 0.056 Day 8 P. falciparum oocysts (Pf Oocysts) directly from whole mosquito lysates. Briefly, the ECL-ELISA is a sandwich ELISA in which immobilized anti-CSP monoclonal antibody (mAb) clone 2A10 captures available Plasmodium CSP antigen. Antibody-bound CSP is subsequently detected and quantified utilizing a biotinylated form of the 2A10 mAb and an avidin-conjugated HRP reporter system. The ECL-ELISA described here obviates the need for dissection of mosquito midguts and salivary glands while simultaneously providing high assay sensitivity and specificity. The performance characteristics and the broad accessibility and ease of use provided by the ECL-ELISA may facilitate the establishment of a comprehensive vector surveillance program and find application as a general diagnostic tool for the detection of other pathologically relevant antigens or host biomarkers of cancer and other disease as well as a critical high throughput validation assay in vaccine development and manufacturing.