Reagents:

Provided by AIDS Vaccine Program (SAIC Frederick, 301-846-1408):

• 96-well plates coated with anti-p24 moAb

• p24 standard (lot SP436H = 9.121 ng/ml, lot SP436I = 6.536 ng/ml, lot SP436J = 7.476 ng/ml, lot SP436K = 7.645 ng/ml)

• primary Ab (rabbit anti-p24 serum)

• secondary Ab (goat anti-rabbit Ig-Peroxidase labeled moAb)

To be provided by user:

• Triton X-100 (Sigma)

• KPL-wash solution (KPL 50-63-00, 800 ml 20X concentrate)

• 10% BSA diluent (KPL 50-61-00, 200 ml)

• 10x PBS (Dulbecco’s without Ca/Mg, BioWhittaker 02732)

• Tween 20 (Sigma P-1379)

• Normal Mouse Serum (Sigma M-9505, 10 ml)

• Normal Goat Serum (GIBCO 16210-015, 100 ml)

• TMB Peroxidase substrate system (KPL 50-76-00, 400 ml)

• H₂SO₄

Solutions:

Lysing solution               =      10% Triton X-100

Wash buffer                   =       1x KPL-wash solution

Sample diluent               =       1% BSA diluent, 1x PBS, 0.2% Tween 20

Primary Ab diluent         =       2% NMS in sample diluent

Secondary Ab diluent     =       5% NGS, 1x PBS, 0.01% Tween 20

Substrate solution           =       TMB Peroxidase system

Stop solution                  =       4 N H₂SO₄

Protocol:

1. Add 1/10 volume of lysing solution to sample. Vortex.

2. Incubate at 37°C for 1 hour.

3. Wash plate with wash buffer, 3x.

4. Make 2-fold serial dilution of standard.

5. Pipette 100 µl of lysed samples and standards into plate.

6. Seal plate and incubate at 37°C for 2 hours or overnight at 4°C.

7. Wash plate with wash buffer, 3x.

8. Add 8 µl of primary Ab to 10.4 ml of primary Ab diluent.

9. Pipette 100 µl into each well.

10. Seal plate and incubate at 37°C for 1 hour.

11. Wash plate with wash buffer, 3x.

12. Add 40 µl of secondary Ab to 12 ml of secondary Ab diluent.

13. Pipette 100 µl into each well.

14. Seal plate and incubate at 37°C for 1 hour.

15. Wash plate with wash buffer, 3x.

16. Pipette 100 µl of substrate solution into each well.

17. Seal plate and incubate at RT for 30 minutes.

18. Add 100 µl of stop solution to each well.

19. Read plate at 450nm. Reference at 650nm.

     

    For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

    Back to Mullins Lab Home Page