Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.2 Plasmodium falciparum Sporozoite ELISAPage 3 of 12Sporozoite ELISA Solutions, Capture and ConjugatePHOSPHATE BUFFERED SALINE (PBS), pH 7.2: Use stock laboratory PBS OR Dulbecco's PBS(Sigma #D5773); adjust pH if necessary. Add 0.01 gm phenol red or 100l of phenol red stock solution(1gm/10ml water)/L PBS. Store 4°C; shelf life is 2 weeks.BLOCKING BUFFER (BB): Shelf life is 1 week at 4°C; BB may be frozen. Use only ELISA grade Sigmacasein.BB – blocking buffer: Use only ELISA grade casein (Sigma C7078). Shelf life is 1 week at 4°C;BB may be frozen.Boiled casein (BB): ½ liter 1 literPBS, pH 7.4 450 ml 900 mlcasein 2.5 g 5.0 g0.1 N NaOH 50 ml 100 mlphenol red* 100 μl 200 μl1. Bring to a boil the 0.1 N NaOH and slowly add the casein (Sigma C7078).2. After casein is dissolved, allow to cool and slowly add the PBS, adjust the pH to ~7.4 with HCl, andadd the phenol red.*A stock solution of phenol red (1g/10ml water) eliminates the need to weigh out small amounts of thismaterial. Phenol red added to BB is only used as a dye to aid in visualization and is optional.Grinding Buffer and Mosquito Sample Preparation: Shelf life at 4°C is 1 week.Grinding solution for approximately one plate:1. Combine 25ml of BB and 125µl of Igepal CA-6302. Mix well to dissolve the Igepal CA-630 in the BB.(Note: IGEPAL CA-630 (Sigma I3021) replaces NONIDET P-40, no longer available from SIGMA-ALDRICH. If available, NONIDET P-40 can be used.)To Grind:1. Place the mosquito, head-thorax only, of no more than 10 pooled mosquitoes in a labeled1.5ml micro centrifuge grinding tube2. Add 50µl of grinding buffer.3. Grind well4. Rinse the pestle with two 100µl volumes of Grinding Solution , catching the rinses in the tubecontaining the mosquito triturate. Final volume will be 250µl. Test or freeze for later use.5. Rinse the pestle in PBS-Tween twice; dry with tissue to prevent contamination betweenmosquitoes.Wash Solution (PBS-Tween): PBS plus 0.05% Tween 20. Add 0.5 ml Tween 20 to 1 liter of PBS. MIXWELL. Store at 4°C. Shelf life is 2 weeks.Monocolonal Antibodies (mAb) Capture and Conjugate: Monoclonal antibodies (mAb) capture andconjugate received from KPL will be lyophilized. Label will list the amount of glycerol water to be added.Glycerol water is a 1:1 mixture of distilled water and glycerol. Glycerol water allows for storage at -20°Cwithout freeze-thawing. This step only needs to be performed when a new vial of capture or conjugateneeds to be reconstituted.