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

Protocol for determination of RT activity blocking antibody titres.

23 serum samples and one sample with known titre 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)
  2 pieces of plastic lids
  

2. Dilute the serum samples.
   The serum samples should be pre-treated according to Sample collection and preparation. When diluting the serum samples, change pipette tips and mix thoroughly between transfers.
• Add 89 µl of Incubation Buffer (F) to wells A1-12 of two sample preparation plates (96-well mictrotiter plates, not supplied).
• Add 90 µl of Incubation Buffer (F) to each of the remaining wells of both plates.
• Add 11 µl of the first serum sample to be analysed to well A1.
• Add 11 µl of the second serum sample to be analysed to well A2 and so on until the serum samples have been added to wells A1-12 of both plates.
• Transfer 10 µl from wells A1-12 down to their corresponding B-wells.
• Transfer 10 µl from wells B1-12 down to their corresponding C-wells and so on until the G-rows of both plates are reached.
• Discard 10 µl from each well in the G-rows of both plates.
  The H-rows are used for controls.

  
  

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.
• Add 20 ml of Incubation Buffer (F) to a 50 ml glass beaker.
• Transfer 200 µl of HIV-1 rRT-standard (D) to the beaker and mix thoroughly.

  
  

4. Start the sample incubation.
• Transfer 90 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 90 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:

  2 PolyA Plates
  2 RT Reaction Components (vial C1)
  1 Reconstitution Buffer (bottle C2)
  2 pieces of adhesive tape
  

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.
• Transfer the contents of both C1 vials to a 50 ml glass 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 both plates.
• Seal the plates with adhesive tape.
• Incubate the plates at 33°C for 20-60 minutes.

  
  

8. Start the RT reaction.
   When transferring start with the H-row and work upwards through the plate as this prevents the need to change pipette tips. The pipette tips should however be changed between plates.
• Take out the PolyA Plates from the incubator.
• Transfer 100 µl from each well of the first sample preparation plate to its corresponding well of the first PolyA Plate.
• Transfer 100 µl from each well of the second sample preparation plate to its corresponding well of the second PolyA Plate.
• Seal the PolyA 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

9. Collect kit components.
• Take out the kit box from the refrigerator and take out the following components:

  1 Concentrated Washing Buffer (bottle E)
  1 RT Product Tracer (vials O)
  1 AP Substrate Tablets (tube P1)
  1 AP Substrate Buffer (bottle P2)
  2 pieces of adhesive tape
  

10. Prepare the wash fluid.
• Add 150 ml of Triton X-100 to 1 litre of distilled water and mix thoroughly with a magnetic spin bar.
  Make sure that the Triton X-100 dissolves completely which takes approximately 10 minutes.
• Add 20 ml of Concentrated Washing Buffer (E) to a 20 litre container.
• Add the dissolved Triton X-100 to the 20 litre container.
• Adjust the volume to 20 litres with distilled water and mix thoroughly.

  
  

11. Prepare the RT Product Tracer.
• Add 12 ml of distilled water to each of the two vials containing RT Product Tracer (O). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Transfer the contents of both O vials to a 50 ml glass beaker.

  
  

12. Stop the RT reaction of the plates.
    After overnight incubation the RT reaction of both plates is stopped by washing. Use approximately 6 ml of wash fluid per well and wash.
• Wash both PolyA Plates twice. Turn the plates 180° between washes if possible.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.

  
  

13. Start the binding of RT Product Tracer.
    When dispensing the tracer, pipette tips should not touch the bottom of the wells.
• Add 100 µl to each well of both PolyA Plates.
• Seal the plates with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plates for 90 minutes at 33°C on an orbital shaker set at gentle agitation.

  
  

14. Prepare the AP substrate solution.
• Add the AP Substrate Tablets (P1) to the AP Substrate Buffer (P2).
• Shake the bottle occasionally and the tablets will dissolve completely in approximately twenty minutes.

  
  

15. Remove excess tracer.
    After 90 minutes of incubation, excess tracer is removed by washing. Use approximately 6 ml of wash fluid per well and wash.
• Wash both PolyA Plates four times. Turn the plates 180° between washes if possible.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.

  
  

16. Start the alkaline phosphatase reaction.
    The AP substrate solution must be mixed thoroughly and equilibrated to room temperature before it is added to the PolyA Plates. When dispensing the substrate solution, pipette tips should not touch the bottom of the wells.
• Add 200 µl of AP substrate solution to each well of both PolyA Plates.
• Cover the plates with plastic lids and incubate them at room temperature under dark cover on an orbital shaker set at gentle agitation.

  
  

17. Read the plates.
    Set the filter of the plate reader to 405 nm.
• Read the absorbance of both plates after 30 minutes of incubation.
• Repeat the reading of the plates at intervals until wells H1-7 give values of approximately 75% of the maximum value within the linear reading range of the plate reader.

  
  

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 (wells H1-7).
• Express the RT activity for wells containing serum dilutions as a percentage of the mean A₄₀₅ HIV-1 rRT standard.
• Plot the percentage of residual RT activity against the serum concentrations for each sample.

The titre of the sample is the serum concentration giving 50% inhibition of the RT activity and is determined with the aid of the obtained graph.




19. Interpret the data.
    Sera taken from HIV infected individuals three months after primary infection normally give titres of 1:1000 - 1:1 000 000. Lower titres, i.e. 1:10-1:1000, are related to sampling shortly after primary infection or possibly due to cross reacting RTb-ab induced by other retroviruses, e.g. HIV-2. Such low titres can also be found in sera taken from individuals in the late stages of AIDS.