BackgroundMalaria is responsible for over 435,000 deaths annually, with most cases occurring in sub-Saharan Africa. Detecting the presence of Plasmodium spp. sporozoites (spzs) in the salivary glands of Anopheles vectors of the parasites using the circumsporozoite enzyme-linked immunosorbent assay (csELISA) is an important malaria surveillance method. The addition of bio-based materials have shown the potential to improve the adsorption and binding of target antigens and thus can improve the sensitivity and detection of analytes in immunoassays. Here, we evaluate the use of two bio-based polymers, chitosan, and cellulose nanocrystals (CNC), as antibody carriers and substrate coating on 96-well plates and on a paper substrate to determine whether detection of Plasmodium falciparum (Pf), P. vivax VK210 (Pv210) and P. vivax VK247 (Pv247) can be improved through assay modification. MethodsModified csELISA assays were conducted using chitosan and CNC as either an antibody carrier or a well coating on 96-well plates (ultra-low- and high-affinity) and results read using standard spectrophotometry of 96-well plates and a quantitative image-based color analysis using photographs of paper plate assays. Changes in frequency and dissipation, resulting from the adsorption of antibodies to model films in a quartz crystal microbalance with dissipation monitoring (QCM-D), were followed to understand better the interactions between the bio-based materials and assay proteins. ResultsThe csELISA performed on high-binding well-plates showed that chitosan, used either as an antibody carrier or well coating, resulted in the greatest increase in detection for Pv210 and Pv247 positive recombinant proteins, increasing absorbance readout values up to 6x for Pv210 and up to 5x for Pv247. On paper csELISA (PcsELISA), chitosan as an antibody carrier yielded the greatest increase in detection sensitivity for all three Plasmodium species when color intensity of positive recombinant proteins was compared to blanks. Compared to controls, chitosan as a carrier resulted in a ~2.5-fold increase in color intensity for Pf, a ~4-fold increase for Pv210, and a ~2-fold increase for Pv247. QCM-D showed a preferred interaction between the assay antibodies and chitosan surfaces, most likely driven by electrostatic interactions. ConclusionThe addition of bio-based materials, mainly chitosan, as shown by QCM-D interactions, absorbance readout values, and image-based color analysis, increased the color intensity of positive samples run through csELISA in all systems, allowing for clearer detection of Plasmodium spp visually, using a spectrophotometer and quantitative color intensity. Furthermore, the adaptation of a PcsELISA coated with chitosan using positive recombinant proteins shows potential as a cost-effective alternative assay platform as it reduced reagent volumes by 80% and assay run time from seven hours to one hour.

Malaria is responsible for over 435,000 deaths annually, mostly occurring in sub-Saharan Africa. Detecting Plasmodium spp. sporozoites (spzs) in the salivary glands of Anopheles (Diptera: Culicidae) vectors with circumsporozoite enzyme-linked immunosorbent assay (csELISA) is an important surveillance method. However, current technological advances are intellectual property and often require of distribution and highly trained users. The transition into paper-based rapid plataforms would allow for decentralization of survillance, especially in areas where it was virtually eliminated. The addition of bio-based materials have shown the potential to improve binding of target antigens, while being widely available. Here, we evaluate the use of chitosan and cellulose nanocrystals (CNC) as antibody carriers and substrate coatings on 96-well plates and on wax hydrophobized paper plates for the detection of Plasmodium falciparum (Pf), P. vivax VK210 (Pv210), and P. vivax VK247 (Pv247). To further improve the user-friendliness of the paper plates a quantitative photograph image-based color analysis was done. Interactions between the materials and the assay antibodies were studied by quartz crystal microbalance with dissipation monitoring (QCM-D). Overall, the addition of chitosan increased the interaction with antibodies and enhanced signaling in all tests. This work demonstrated that the adaptation of a PcsELISA shows potential as a cost-effective alternative assay platform easily adaptable in deployable testing sites that also showed reduction in reagent volumes by 80% and assay run time by seventh. While dipstick assays were previously developed, paper-based assays are a cost-effective and field-deployable alternative, reducing volumes of reagents that could be used in malaria control and elimination settings.