Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.2 Plasmodium falciparum Sporozoite ELISAPage 4 of 12Notes and Troubleshooting for Sporozoite ELISAs1. Do not add sodium azide to solutions as it is a peroxidase inhibitor. We no longer add thimerosal tothe solutions, as this is mercury-based and presents problems with proper disposal.2. To fill each of the 96 wells on a plate with 50μl requires 4.8ml. It is convenient to make up 5.0ml ofeach mAb solution and 10.0ml of substrate (100µl/well) per plate.Species mAb g/50μl/WELL g/5ml l STOCK/5ml PBSPf capture 0.200 20.0 40Pv - 210 capture 0.025 2.5 5Pv - 247 capture 0.025 2.5 5Pf peroxidase 0.050 5.0 10Pv - 210 peroxidase 0.050 5.0 10Pv - 247 peroxidase 0.050 5.0 10l STOCK/5ml BB3. Cover plate during incubations to prevent evaporation. Incubate plate in the dark (especially forincubation of peroxidase conjugate and substrate solution). An efficient way of doing this is to use asmall cardboard box lid or to line the lid of a pipette tip box with aluminum foil.4. ELISAs can be carried out on fresh, frozen or dried mosquitoes. If specimens are to be dried, theymust be processed quickly and kept dry (store with desiccant) to prevent microbial growth that canresult in high background values in the ELISAs. Once identified to species, mosquitoes should bepooled in groups of maximum 10 and stored at -20°C or dried over silica gel until they can beprocessed. Store the triturate at -20°C until tests are to be performed.5. Negative controls: Triturate laboratory reared, known uninfected female mosquitoes (same as testspecies if possible) in 50μl BB:IG-630, dilute with 150μl BB (total volume 200μl) and place 50μl fromeach into negative control wells 1B-1H for initial testing or wells 1A-1H for conformational testing. Todetermine the negative cut off value for initial testing calculate mean on these 7 negative controls andretest any mosquito with a value two times the mean.6. Phenol red added to BB is only used as a dye to aid in visualization and is optional.7. Only high, lab grade paper towels should be used while performing CS-ELISA testing, particularly to“bang” the ELISA plate on, to remove excess solutions. Use of brown paper towels and kitchenpaper towels should be avoided as the result is high background values. It is thought that fibers fromthese towels adhere to the plates and interfere with detection. If high, lab grade paper towels are notavailable, perform this step over the sink or ensure careful removal of all solutions with a vacuumsystem for each required step.8. Do not vortex samples as this can lead to high background and inaccurate absorbance values. Shortcentrifugation can but used to settle body parts and allow pipetting of a clean sample. Thiscentrifugation will not pellet sporozoites and they will remain suspended in the supernantant.9. Wipe the bottom of the ELISA plate with alcohol to remove debris and oils from ELISA plate beforereading. Fingerprints, oils and debris on the plate can lead to inaccurate absorbance values.10. BB should be placed in wells without negative mosquito controls, positive controls, or samples as“filler”.11. The Corning® 96 well clear round bottom PVS (soft plate) cannot be reliably used with certain,especially newer, models of plate readers. This is due to distortion of the plate upon entering thereader carriage or a poor fit of plate in the reader carriage. This can be solved by using Costar 96-Well EIA/RIA plates (Round well; high binding), available from www.fishersci.com see supplies list).