Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.2 Plasmodium falciparum Sporozoite ELISAPage 6 of 122. The highest concentration to be used on ELISA plate is prepared ahead of time. When addition ofpositive controls to wells of ELISA plate is required, serial dilutions are made directly on the plate.This requires very accurate and advanced pipetting skills.Positive controls (be sure to label all vials):P. falciparum:STOCK: Add 1000l BB to lyophilized positive control (5g) (do not use glycerol:water or wateralone) to give 10,000pg/l BB.Vial I = Transfer 20l (200,000pg) of STOCK to 1000l BB to for 100pg/l BB.Vial II = Transfer 10l (1000pg) of Vial I to 500l BB for 2pg/l or 100 pg/50l BBBegin in well 2A as wells 1A-1H should be reserved for 8 negative control mosquitoes whenretesting. Freeze the stock solution and Vials I and II for continued use.Use 100µl Vial II in well 2A and add 50l BB to wells 2B through 2H. Pipette 50l from well 2Aand add to well 2B. Continue this serial dilution through well 2H and discard 50l from well 2H soeach well contains 50l of BB + diluted positive control.Concentrations of positive controls are 100, 50, 25, 12, 6, 3, 1.5 and 0pg/50l BB (starting with2A and finishing with 2H)Run standard curve in triplicate (wells 2A-4A to 2H-4H) as well as 8 negative control mosquitoes(wells 1A-1H) when retesting.P. vivax-210:STOCK = Add 500l BB to lyophilized positive control (5g) (do not use glycerol:water or wateralone) to give 10,000pg/l BB.Vial A = Transfer 10l (100,000pg) of STOCK to 1,000l BB for 100pg/l BB.Vial B = Transfer 20l (2,000pg) of Vial A to 500l BB for 4pg/l BB.Vial C = Transfer 200l (800pg) of Vial B to 800l BB for 0.4pg/l or 40 pg/50l BB.Begin in well 2A as wells 1A-1H should be reserved for 8 negative control mosquitoes whenretesting. Freeze the stock solution and Vials I and II for continued use.Use 100µl Vial C in well 2A and add 50l BB to wells 2B through 2H. Pipette 50l from well 2Aand add to well 2B. Continue this serial dilution through well 2H and discard 50l from well 2H soeach well contains 50l of BB + diluted positive control.Concentrations of positive controls are 40, 20, 10, 5, 2.5, 1.25, 0.6 and 0pg/50l BB (starting with2A and finishing with 2H)Run standard curve in triplicate (wells 2A-4A to 2H-4H) as well as 8 negative control mosquitoes(wells 1A-1H) when retesting.P. vivax-247:STOCK = Add 1000 µl BB to lyophilized positive control (4.55ug) (do not use glycerol:water orwater alone) to give 4,550pg/l BB .Vial 1 = Transfer 20l (91,000pg) of STOCK to 1,000l BB to give 4,550pg/l or BB.Begin in well 2A as wells 1A-1H should be reserved for 8 negative control mosquitoes whenretesting. Freeze the stock solution and Vials I and II for continued use.Use 100µl Vial 2 in well 2A and add 50l BB to wells 2B through 2H. Pipette 50l from well 2Aand add to well 2B. Continue this serial dilution through well 2H and discard 50l from well 2H soeach well contains 50l of BB + diluted positive control.Concentrations of positive controls are 4450, 2275, 1140, 570, 285, 140, 70 and 0pg/50l BB(starting with 2A and finishing with 2H)Run standard curve in triplicate (wells 2A-4A to 2H-4H) as well as 8 negative control mosquitoes(wells 1A-1H) when retesting.