Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance Monitoring5.2 Microplate Enzyme Activity Assays5.2.1 Microplate Insensitive Acetylcholinesterase AssayPage 1 of 25.2.1 Microplate Insensitive Acetylcholinesterase AssayWilliam G. BrogdonIntroductionInsensitive acetylcholinesterase (AChE) has been associated with resistance to carbamates andorganophosphates. Propoxur is used in this assay to inhibit the activity of the sensitive (i.e. susceptible)AChE, allowing the detection of the altered enzyme when it is present: The number of alleles ofinsensitive AChE is greater as the yellow color darkens. It may be possible to clearly distinguishhomozygous resistant, heterozygous and homozygous susceptible individuals by their discreteabsorbance classes.Materials• Acetone• Acetylthiocholine iodide (ATCH)• Dithiobis (2-nitrobenzoic acid), (DTNB)• 0.25 M KPO 4 buffer prepared as described in Microplate Enzyme Assays Introduction• Propoxur, technical gradeReagent preparationATCH1. Dissolve 75 mg acetylthiocholine iodide (ATCH) and 21 mg propoxur in 10 ml acetone.2. Add 90 ml 0.25 M KPO 4 buffer.3. Store at 4° C for up to 3-4 days.DTNB (Ellman’s reagent)1. Dissolve 13 mg Dithiobis (2-nitrobenzoic acid), (DTNB), in 100 ml 0.25 M KPO 4 buffer.2. Store at 4° C for up to 3-4 days.Protocol1. To the plate containing the mosquito homogenates (see Microplate Enzyme Assays Introduction),add 100 µl of KPO 4 to the negative control wells.2. Add 100 µl ATCH to each well.3. Add 100 µl DTNB to each well.4. Read plate immediately (T 0 ) with microplate reader at 414 nm.5. Read plate at 10 minutes (T 10 ).6. Subtract the T 0 reading from the T 10 reading and use this for your statistical analyses.