Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance Monitoring5.2 Microplate Enzyme Activity AssaysPage 1 of 45.2 Introduction to Microplate Enzyme Activity AssaysWilliam G. BrogdonIntroductionIn this section, preparation of the mosquito homogenates for analysis and data analysis are described.Specific assays are presented in the following sections.The presence of enzyme activities relevant to insecticide-resistance is often performed using microplateassays. These can be conduced with either pupae or adults. Since mosquitoes must be destroyed, itshould only be used to sample the population to determine if a specific mechanism is present and at whatfrequency it occurs. This method does, however, allow you to detect underlying resistance mechanismsthat may not be detected using bioassays. A disadvantage of these assays is that there is no way toreliably detect heterozygotes for many enzymes. These assays should always be performed with asusceptible control so baseline values can be used for comparison. Suitable samples can be obtainedfrom among the MR4-held stocks.Specimens for biochemical analysis must befreshly killed and immediately used or frozenfor later analysis. No chemicals of any sortshould be used for killing since they caninterfere with the assays. Samples may befrozen a stored a -20 o C for up to 7 days.Thereafter, -80 o C or colder storage isadvised. Samples may be stored only brieflyin the refrigerator.Alcohol-persevered specimens cannot beused for biochemical analysis. The alcoholwill reduce or eliminate the enzyme activitiesthat these procedures are measuring.Figure 5.2.1. An example of an oxidase enzymebioassay with elevated enzyme levels (possibleresistance) indicated by the darker colors.You may wish to perform total solubleprotein analysis on your samples. Thisenables size-correction when comparingdifferent species or different "broods" of thesame species which allows you to correctfor higher enzyme levels due only to theirsize. The standard curve is usuallydetermined using bovine serum albumin.Procedures for how to do this are provided by the manufacturer of the protein detection system used orare widely available and will not be discussed further.Collecting and interpreting dataA plate-reading spectrophotometer will be used to collect data at the appropriate absorbing wavelength(described in the protocol for each assay). The software for these instruments will probably include anumber of data handling features that may be especially useful.Following is a brief introduction to data analysis. A susceptible population shows an upper absorbancerange limit for susceptibility in terms of activity. Individuals with levels above that threshold are lesssusceptible (Figure 5.2.2).The susceptible population (white bars) is normally distributed with regard to enzyme activity. The upperrange limit at e.g. 570 nm (in this esterase assay) is 0.9. This becomes the resistance threshold. Thepopulation shown in black is a hypothetical field population containing individuals with elevated activity