Adaptation of ELISA detection of Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in mosquitoes to a multiplex bead-based immunoassay

Abstract

Background Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites. Methods Recombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. Results Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8–4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined. Conclusions The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

Evidence for a role of Anopheles stephensi in the spread of drug- and diagnosis-resistant malaria in Africa

“…Part of the homogenate (50 µl) was used for nucleic acid extraction using cetyl trimethyl ammonium bromide 62 ; 100 µl grinding buffer (0.5% w/v case in, 0.1 N NaOH in 10 mM PBS, pH 7.4, and 0.5% IGPAL CA-630) was added to the remaining that was used to screen samples for circumsporozoite in bead-based assay. Circumsporozoite bead-based assay : We adopted the most advanced (highly sensitive) bead-based assay for infection detection in mosquitoes 66 by targeting CSP. Antibody-coupled magnetic beads and biotinylated secondary antibodies were obtained from the Centers for Disease Control and Prevention, Division of Parasitic Diseases and Malaria, Entomology Branch, Atlanta, GA, USA, and implemented as described before 7 and were run using MagPix immunoanalyzer (Luminex Corp, CN-0269-01).…”

Rapid production of Plasmodium sporozoite detection paper dipstick assays using cellulose nanocrystals: Proof‐of‐concept for bio‐based, locally developed, point‐of‐care devices

“…In addition, results could be obtained without the use of an extra layer such as the BSA used in the controls. Furthermore, only 16.7 ng of antibody were needed for each test, which is lower than the 100‐µg required for each well in csELISA [ 22 ] and an improvement compared to the commercial dipstick assays for sporozoite detection. [ 10 ] The test showed high sensitivity as the saturation of the ink dot can be observed with a black color in the test strip when a 5 ng mL ‐1 antigen‐PBS solution was used.…”

Assessment of Bio-Based Materials as a Sustainable and Scalable Alternative for Detection of Plasmodium spp. (Haemospororida: Plasmodiidae) Sporozoites in Field Deployable Testing

“…MRA-890; MRA-1028K) (Malaria Research and Reference Reagent Resource Center (MR4), 2017). These recombinant proteins have been used in other studies adapting the csELISA to other assay platforms, such as multiplex bead-based immunoassays (Wirtz et al 1985, Sutcliffe et al 2021). The P. falciparum positive control antigen is derived from recombinant protein R32tet 32 produced in Escherichia coli (Smith Kline and French Laboratories, Philadelphia, PA) (Young et al 1985).…”