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Measuring HIV-1 viral concentration in the supernatant of cultures by p24Gag ELISA
Author: Marielle Cavrois, updated date: , view: 45, Q&A: 0
Tags: HIV and Productive Infection

Procedure

Measuring HIV-1 viral concentration in the supernatant of cultures by p24Gag ELISA

Notes:

  • The  best quantification is achieved when diluting HIV-1 containing viral supernatant in PBS 2% FBS 0.5% triton. This ensure that virus gets lysed  before proceeding to dilution

  • Supernatant from 293T transfected with HIV-1 provirus should give a concentration between 200 ng/ml and 1000 ng/ml of p24Gag. Supernatant from  infected SupT1 cells can produce up to 100ng/ml of p24Gag

  • The Standard curve goes from 0.4 ng/ml to 6.25 pg/ml. So, dilute the supernatant and take several dilution that could fall on the curve

  • Determine the number of raws of wells that will be needed

 Materials and Reagents


  • Perklin Elmer/NEN Life Science Products HIV-1 p24 ELISA

  • PBS

  • FBS

  • 5% Triton

Equipment

  • Plate reader: ELISA microtiter plate reader capable of measuring absorbance at 490 nm or 492 nm and > 600nm filter      capability

  • Incubator at 37˚C

  • 96 well plate washer (ideally)

  • Multi channel pipetman

  • 96 w plate to prepare dilutions

Software

  • Excel

Procedure

1.     Prepare Solutions

a.     Prepare the washing solution by diluting the 20X concentrate into appropriate amount of water

b.     Prepare 50 ml of PBS containing 2% FBS and 0.5% Triton X-100 final

2.     Dilute the standard p24 using the standard p24 and diluent (200 µl will be needed)

a.     20 µl of standard into 1000 µl of PBS containing 2% FBS and 0.5% Triton X-100

b.     200 µl of (a) into 1000 µl

c.     500µlof (b) into 1000 µl final: 400pg/ml

d.     500µlof (c) into 1000µlfinal: 200pg/ml

e.     500µlof (d) into 1000µlfinal: 100pg/ml

f.      500µlof (e) into 1000µlfinal: 50pg/ml

g.     500µlof (f) into 1000µlfinal: 25pg/ml

h.     500µlof (g) into 1000µlfinal: 12.5pg/ml

i.       500µlof (h) into 1000µlfinal: 6.25 pg/ml

3.     Dilute out viral supernatant in PBS containing 2% FBS and 0.5% Triton X-100 (do step by step 1/10 step dilutions)

a.     For 293T produced virus, test dilutions 1/10,000; 1/100,000

b.     For Supernatant, test dilutions 1/100; 1/1,000

4.     Take the elisa plate from the kit, remove the raws that will not be used (keep in the bag at 4˚C). Transfer 200 µl 2% FBS and 0.5% Triton X-100 final in first well for negative control, then load the remaining 7 wells with the standards (200 µl each).  Finally transfer 200 µl of samples to be measured (ie the different dilution of the viral supernatant with unknown concentration of p24Gag).

5.     Incubate 60 minutes at 37oC. Warm the detector antibody to room temperature during this incubation. 

6.     Wash in plate washer (choose numbers of rows, 5 cycles, prime first).  Make sure all liquid is removed.

7.     Add 100 µl detector antibody. 

8.     Incubate 60 minutes at 37oC.

9.     Wash in plate washer (choose numbers of rows, 5 cycles, prime first).  Make sure all liquid is removed.

10.  While washing, dilute the SA-HRP @ 1:100 in Streptavidin-HRP diluent.  Make enough for 100 µl/well plus a little extra.

11.  Add 100 µl diluted SA-HRP to wells.  Incubate 15 minutes at room temperature.  Wash plate.

12.  While washing plate, dissolve OPD tablet in 11ml of Substrate Diluent.  Add 100 µlt o each well.  Incubate at room temperature until desired reading is reached (~5-15 minutes).

13.  Add 100 µl Stop solution and read on plate reader.

 

Data analysis

Determine the titration curve best fit and obtain the equation of the curve/line. Calculate the concentration of diluted unknown and determine the concentration of the unknow undiluted


References

https://www.hanc.info/labs/labresources/procedures/ACTGIMPAACT%20Lab%20Manual/Perkin-Elmer%20(NEN%20Life%20Sciences)%20Hiv-1%20p24%20Antigen%20ELISA.pdf


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