The RetroSys™ RT Activity Kit
Product code: RT-001

Protocol for screening for RT activity blocking antibody.

84 serum samples set in duplicate may be analysed with one kit.

1. Collect kit components.
• Take out the following components from the refrigerator:

  1 HIV-1 rRT-standard (vial D)
  1 Incubation Buffer (bottle F)
  1 RTb-ab Negative Reference (tube G)
  2 pieces of adhesive tape
  2 pieces of plastic lids
  

2. Dilute the samples.
   The samples should be pre-treated according to Sample collection and preparation.
• Add 10 µl of the first serum sample to be analysed to well A1 of two sample preparation plates (96-well mictrotiter plate, not supplied).
• Add 10 µl of the second serum sample to be analysed to well A2 and so on until the serum samples have been added to wells A1-G12 of both plates.
• Add 10 µl of RTb-ab Negative Reference (G) to wells H1-12 of both plates.

  
  

3. Dilute the HIV-1 rRT-standard.
• Add 7.5 ml of Incubation Buffer (F) to the vial containing lyophilised HIV-1 rRT- standard (D). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Add 38 ml of Incubation Buffer (F) to a glass beaker.
• Transfer 200 µl of HIV-1 rRT-standard (D) to the beaker and mix thoroughly.

  
  

4. Start the sample incubation.
• Add 170 µl of standard enzyme (beaker) to all wells except H8-12 of both sample preparation plates.
  Remove the beaker from the working area. This beaker should not be used any further in this assay.
• Add 170 µl of Incubation Buffer (F) to wells H8-12 of both plates.
• Cover the plates with plastic lids and incubate them at 33°C for 90 minutes on an orbital shaker set at gentle agitation.

  
  

5. Collect kit components.
• Take out the following components from the refrigerator:

  PolyA Plates
  RT Reaction Components (vial C1)
  Reconstitution Buffer (bottle C2)
  

6. Prepare the reaction mixture.
• Add 12 ml of Reconstitution Buffer (C2) to each of the two vials containing RT Reaction Components (C1). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Transfer the contents of both C1 vials to the beaker.

  
  

7. Prepare the PolyA Plates.
• Take out both PolyA Plates from their foil pouches.
• Add 100 µl of reaction mixture (beaker) to each well of the plates.
• Seal the plates with adhesive tape.
• Incubate the plates at 33°C for 20-60 minutes.

  
  

8. Start the RT reaction.
   When transferring samples, change pipette tips between each transfer.
• Take out the PolyA Plates from the incubator.
• Transfer 100 µl from each well of the first sample preparation plate to its corresponding well in the first PolyA Plate.
• Transfer 100 µl from each well of the second sample preparation plate to its corresponding well in the second PolyA Plate.
• Seal the plates with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plates overnight at 33°C on an orbital shaker set at gentle agitation.
  

  
  

  
  

Day two

Follow the procedures as described in C₁. Protocol for determination of RT activity blocking antibody titres, steps 9-17.

   18. Process the data.

Use an alkaline phosphatase reading time at which wells H1-7 give approximately 75% of the maximum value within the linear measuring range of the plate reader. Follow the instructions for each plate separately.

• Calculate the mean A₄₀₅ of the background controls (wells H8-12).
• Subtract the mean A₄₀₅ of the background controls from the obtained A₄₀₅ of the other wells.
• Calculate the mean A₄₀₅ of the HIV-1 rRT standard incubated with the RTb-ab negative reference (wells H1-7).
• Express the RT activity for wells containing serum samples as a percentage of the mean A₄₀₅ HIV-1 rRT standard incubated with the RTb-ab negative reference.
• Compare the percentage of residual RT activity obtained from the two plates for each serum sample. If they are similar then calculate a mean value. If they are significantly different then re-analyse the serum sample.
  

  19. Interpret the data.

Strong RTb-ab positive serum samples leave less than 25% residual RT activity compared to the negative controls. Serum samples that leave 50% residual RT activity or less are considered RT-blocking. However, as for all HIV-1 ab assays, sera from persons not known to be HIV-1 infected, should be verified by an alternative HIV-1 assay. In addition it is recommended for serum samples leaving 30-50% residual RT activity to perform an analysis for HIV-2, as weak cross-reactivity between HIV-1 and HIV-2 occurs.