The RetroSys™ RT Activity Kit
Product code: RT-001
Applications described.
A. Quantification of RT activity.B. Screening for RT activity in cell supernatants.
C. Determination of RT activity blocking antibody.
D. Determination of IC50 value of RT activity inhibiting substances.
A. Quantification of RT activity.
When using crude samples RT activity inhibitory factors may be present. In order to ensure a quantitative determination of enzyme activity in such samples, it is important to establish that the enzyme reaction is linear with time. It is also important to verify that the measured RT activity is proportional to the concentration of the sample in the assay. Thus samples to be quantified should be analysed at several dilutions as well as at different RT reaction times. The measurement should be carried out on serially diluted samples set on two plates. For one of the plates the RT reaction is stopped by washing after three hours of incubation, while the other is incubated overnight.
On day two the plates are incubated with the RT Product Tracer. After removing excess tracer by washing the plates, the amount of bound tracer is determined by an alkaline phosphatase / pNPP colour reaction measured at different times. The level of RT activity in the sample is calculated from the HIV-1 rRT standard dilutions set on each plate.
The measuring range of the assay is the activity of 0.01-50 pg recombinant HIV-1 RT per well. The lower detection limit corresponds to the presence of 1 pg/ml of active RT in undiluted cell supernatant, if the analysis is performed with the cell supernatant diluted 1/5, which is the highest concentration that can be used without major disturbances (Ekstrand et al 1996).
For comparison the activity of 1 pg of the supplied HIV-1 rRT- standard
is equal to the activity of 4 µU of the rRT from Boehringer Mannheim.
B. Screening for RT activity in cell supernatants.
When processing large series of samples, of which only a few are expected to be RT positive, it may be advantageous to perform a semi quantitative screening. For instance when analysing cell supernatants from virus isolation cell cultures for presence of HIV.
Screening differs from quantification in that a small amount of crude undiluted sample is used. The small sample amount is necessary in order to avoid disturbances. As a result a larger number of samples can be analysed per kit. In addition sample handling becomes more convenient. However, it also means that the level of accuracy in measuring RT activity is reduced.
In all other relevant aspects this application follows the same procedure
as for quantification of RT activity. The assay is performed by using
two incubation times in the RT reaction step, three hours and overnight.
C. Determination of RT activity blocking antibody.
The sensitivity of an assay for RTb-ab depends on the amount of RT used. Since the RetroSys™ assay uses a small amount of RT and overnight polymerisation reaction, it gives a very high sensitivity for RTb-ab detection and measures a broad range of titres. When monitoring titre changes it is advised to include the previous sample from the individual together with the new one that is to be analysed. This is to authenticate the significance of any changes in titre.
Each serum is analysed using seven tenfold serial dilutions. A standardised small amount of recombinant HIV-1 RT is mixed with each serum dilution. RTb-ab negative reference and background controls are included. The samples are incubated for 90 minutes in order to let the antibodies bind the enzyme. After the incubation the remaining RT activity in each sample is determined by starting the RT reaction. For both plates the RT reaction is stopped by washing after overnight incubation.
Only the samples containing unblocked RT have now synthesised a DNA strand containing BrdUMP. The product is quantified by the addition of the RT Product Tracer to all wells. After 90 minutes of incubation, excess tracer is removed and the amount of bound tracer is determined by an alkaline phosphatase / pNPP colour reaction measured at different times. The measured residual RT activity for each serum dilution is corrected for background signal and then calculated as a percentage of the RT activity in the RTb-ab negative reference. The titre of RTb-ab in the serum is expressed as the dilution at which the serum inhibits 50% of the RT activity.
The kit can be used to determine RTb-ab in 23 sera and one sample with known titre. Titres in the range of 1:1 to 1:1 000 000 or greater, are detected using the protocol given. The lowest amount of RTb-ab detected, i.e. the titre 1:1, corresponds to the blocking of 50% of the activity of 2.4 pg of HIV-1 rRT by incubation with 8 µl of undiluted serum.
The kit can also be used to screen sera for HIV-1 RTb-ab presence. In
this case 84 sera can be analysed in duplicate using an amount corresponding
to 8 µl of undiluted serum.
D. Determination of IC50 value of RT activity inhibiting substances.
The RetroSys™ RT activity kit can be used to study RT inhibiting substances. In order to obtain optimal sensitivity the analysis should be done in a system where the concentrations of substrate and primer are close to the Km value.
When determining IC50 values the substances that are to be analysed are serially diluted. The diluted substances are then added to a plate with reaction mixture. After 30 minutes of preincubation at 33 C, the reaction is started by the addition of a standardised amount of RT. The RT will now incorporate BrdUMP depending on the level of inhibition.
The reaction is stopped by washing the plate. The product is quantified by the addition of the RT Product Tracer which binds to the incorporated BrdUMP. After removing excess tracer the amount of bound tracer is determined by an alkaline phosphatase / pNPP colour reaction.
After correction for background signal, the measured residual RT activity for each substance dilution is calculated as a percentage of the measured RT activity in absence of inhibiting substances. The inhibitory effect of each substance is expressed as an IC50 value i.e. the concentration at which 50 % of the RT activity is inhibited.
The kit can also be used for screening a larger number of substances for inhibitory effects. In this case the substance is not serially diluted but used at one concentration. A protocol for this is not provided.
It should be noted that this test is not sensitive to all kinds of inhibitors.
For instance, inhibition by substances competing with dNTPs other than
dTTP are not detected with this assay as it is based on the use of prA/odT
as template/primer.
