Simulation of HIV-1 (AIDS) Detection using
ELISA Assays
Objectives:


To understand the molecular biology of human immunodeficiency virus
(HIV) and the pathogenesis of acquired immune deficiency syndrome
(AIDS)
Learn the experimental concepts and methodology involved with
enzyme linked immunosorbent (ELISA) assays in the context of
clinical screening of serum samples for antibodies to the virus.
Materials Needed:
Microtiter plate containing 96 wells (2 students/plate)
Gloves (1 pair per student)
Safety goggles
Pipets (11)
Marker
Paper towels
Microtubes Required with Labels:
+ Control
1
Primary Antibody
1
– Control
1
Secondary Antibody
1
Donor #1
1
Substrate
4
serum
Donor #2
1
Antigen (HIV)
1
serum
1
Procedures:
1. Divide microtiter plate into 2 halves length wise, labeling rows by
number 1-4. Then divide by every 3 wells across, drawing a dividing
line. You will perform each test in triplicate to give a meaningful
number for viable results.
1. Control +
2. Control –
3. Donor 1
4. Donor 2
2. Coat all wells being used with antigen (HIV antigen) ~ 2 drops per
well, using correctly labeled pipet.
3. Incubate 2 minutes at room temperature.
4. Discard antigen from plate on to paper towels and tap dry.
5. Add 2 drops per well of the primary antibody to all wells being used,
use correctly labeled pipet.
6. Incubate for 2 minutes at room temperature
7. Add 2 drops per well of the secondary antibody (Rabbit  Human
IgG/HRP) to all wells being used with correctly labeled pipet.
2
8. Incubate for 2 minutes at room temperature
9. Add 2 drops per well of the Control +, Control –, Donor 1, and Donor 2
using correctly labeled pipet.
10. Add 1 drop per well of the substrate to each row, using different
pipets for each row.
11. Record results
3
Instructor’s Guide:
Prepare experiment reagents as follows (be sure to label clearly each for
students use)
+ Control – NaOH (25% ––2.5g in 100 ml of H2O)
– Control – H2O
Donor #1 serum – H2O
Donor #2 serum – NaOH
1 Antibodies ~ 50 ml
Antigen –50 ml H20
Secondary antibody – Rabbit  Human IgG/HRP (Horse Radish Peroxidase) –
(H2O)
Substrate – 50 ml Phenolphthalein
Avoiding Common Errors:
 Students should be advised to be careful when transferring solutions
into and out of microtiter plates.
 Be careful to use clean and appropriately labeled pipets and avoid
contamination of adjacent wells.
4