Immunodot assay of Plasmodium falciparum sporozoites in mosquitoes using a direct binding membrane system

We describe a membrane based immunodot assay for the detection of Plasmodium falciparum sporozoites mixed with mosquitoes. A crude sodium dodecyl sulfate extract of mosquitoes and sporozoites is passed through a bi-layered membrane system, the top layer being a polyvinyldiene difluoride hydrophilic pre-filter which screens out debris but allows the passage of antigen. Sporozoite, as well as mosquito, proteins are bound to the hydrophobic membrane below. This membrane was probed with a monoclonal antibody to the repeat region of the P. falciparum circumsporozoite protein, a peroxidase labeled second antibody and a tetramethyl-benzidine substrate. The method detects as few as 10 sporozoites/mosquito or 100 sporozoites in a pool of 10.