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

• 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.

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.