THE HRP2 ASSAY FREQUENTLY ASKED QUESTIONS (FAQ)
Last updated 19.10.2004
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WHAT DOES A TYPICAL ELISA RESULT LOOK LIKE?
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Typically (with both the CELISA test kits and the in-house ELISA) we get OD values for controls of 1.2 to 2.0. The ODs at full growth inhibition ("background") are around 0.2 to 0.4. Always try to keep your dose-response curve within optimal testing range of the ELISA (dilute if your background is too high).
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TROUBLESHOOTING: HIGH BACKGROUND
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Essentially there are 2 likely reasons for a high background (e.g. if your positive controls are around OD 1.5 and your background (i.e. OD at full inhibition) is >0.7). 1.) High parasite density: if the parasite density in your sample and therefore the HRP2 level is too high then the background will also be high, even if the culture went perfectly well. Solution: Further dilute sample. In most cases it will not be necessary to repeat the culture. 2.) The ELISA is too sensitive: Some of the recently (2004) shipped CELISA test kits from CELLABS are obviously more sensitive and therefore require higher dilutions of the sample. Solution: Further dilute sample. In most cases it will not be necessary to repeat the culture.
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TROUBLESHOOTING: LOW OD IN CONTROLS
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Essentially there are 2 likely reasons for a low readings in controls (e.g. if your positive controls are considerably below OD 0.8). 1.) Low parasite density: if the parasite density in your sample is low the controls will also be low. In this case the background will also be very low (e.g. <0.2). From our experience with fresh field isolates parasite densities as low as 0.01% (at 1.5% hct) still produce good results. Below 0.01% testing may result in inadequate dose-response curves 2.) Inadequate growth in culture: In this case the background will be normal (e.g. control = OD 0.5, background = OD 0.4). Recheck your culture conditions. From our experience approximately 80% of fresh field isolates should give adequate growth in a 72 hr culture.
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48 VS. 72 HOUR INCUBATION
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Longer incubation times (72 hours) have the major advantage that they better imitate physiological conditions, that they generally lead to a clearer increase in parasitemia and biomass (and therefore also in HRP2), that they allow for the testing of slow acting drugs (such as antibiotics) without changes in the protocol, and that the success rate will be considerably higher. We therefore exclusively use a 72 hour incubation time.
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WHY HRP2?
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HRP2 is one of the best documented malaria proteins and a good indicator of parasite growth (and its inhibition by antimalarial drugs). However, its main advantage lies in the availability of monoclonal antibodies and especially in the availability of commercial ELISA test kits, which significantly simplifies the process of drug sensitivity testing.
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FRESH VS. CULTURE ADAPTED PARASITES
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The major difference between field and culture-adapted parasites is the fact that field samples come at varying parasite densities (sometimes below 0.05%) and that dilution may be difficult, particularly when working in the field, whereas culture adapted Pf strains can easily be adjusted to any parasite density. We therefore use two different SOPs for field and lab work.
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BACKGROUND
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As HRP2 is a relatively stable protein, all tests lead to a naturally high background due to preexisting HRP2 (which may easily be eliminated). In addition the test measures parasite growth over the whole test period. Very slow acting drugs may therefore lead to a further increase in background HRP2. Use well developed rings (not very early rings) for testing to reduce background for slow acting drugs. To completely exclude the background draw a culture sample (e.g. from control wells incubated on a separate drug free 96-well plate) after 24 hours and subtract this value from all results. This will restrict measurement of parasite growth to the later part of the culture (similar to the isotopic assay).
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PARASITE STAGE
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Similar to other drug sensitivity assays (such as the isotopic assay) the majority (>80%) of the parasites should be in ring stage when starting the assay. Preferably start the testing when the rings are around 12 hours old (well developed cytoplasm and vacuole). Very early rings (0 to 6 hours after invasion) are not as well suited for testing as they usually do not grow as well in culture and may result in a higher background.
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PARASITE DENSITIES
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From our experience the assay can easily be performed with an initial parasitemia as low as 0.01% (at 1.5% hct). The range yielding the best results lies between 0.02 and 0.07%. Samples with an initial parasitemia of more than 0.1% need to be diluted accordingly, either before incubation (with uninfected RBC) or before performing the ELISA (with RPMI 1640 or distilled water).
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AMOUNT OF CELL MEDIUM MIXTURE USED IN THE ASSAY
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Two hundred uL of cell medium mixture and 25 uL of drug solution per well were used throughout our study. Since the ELISA requires only 100 uL sample volume, the assay can equally be performed with a reduced amount of cell medium mixture (remember to adjust your drug concentrations accordingly).
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ASSESSMENT OF PREEXISTING HRP2
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A sample of the cell medium mixture may be set aside and frozen before the start of the incubation to assess preexisting HRP2. This sample is tested in the course of the drug sensitivity test and its OD subtracted from all results. This allows for a very precise estimation of the rise in HRP2 levels. If full inhibition of parasite growth can be expected, however, it will be sufficient to use the lowest value in each test instead.
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HANDLING OF RAW DATA
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There is essentially two different approaches to the handling of the raw data. In a simplified model the raw data obtained from the ELISA plate reader (optical density values) may directly be used to calculate effective concentrations (e.g. using the software from this website). In a more advanced model the optical density (OD) values may be transformed into percentages of HRP2 by using a standard curve. Most modern ELISA plate readers allow for a relatively simple calculation of standard curves. However, this requires a serial dilution of the control (or standard) to be tested in the ELISA.
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USE OF PLATES PREDOSED WITH ANTIMALARIAL DRUGS (e.g. from WHO)
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The plates we use are dosed with drugs just before starting the culture. However, predosed plates, such as those provided by WHO for the morphological assay, may equally be employed (Remember to adjust your concentrations to the amount of cell medium mixture used).
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Drug Resistance
HRP2
