1. EDVOKIT#271: Simulation of HIV-1 Detection: ELISA
Lab 24 Goals and Objectives:
EDVOKIT#271: Simulation of HIV-1 Detection: ELISA
and Exercise 74: Enzyme Linked Immunosorbent Assay
Work in pairs: ten groups possible
Perform as indicated in supplement pg
Each group will need:
Micropipettor ( ) and tips
5 transfer pipettes
Beaker for waste solution and tips
Microtiter plate
Reagents: PBS, +, DS1, DS2
2°Ab (Secondary antibody: rabbit α human) Substrate: will be provided at time it is needed
Exercise 72: Slide Agglutination (Indirect/Latex) Test
Perform yourself at front of room or observe demo

2. Indirect HIV ELISA
1. bind HIV antigen to well: protein adheres to plastic nonspecifically
2. wash off unbound antigen:
PBS = phosphate buffered saline: maintains pH and salinity to prevent protein denaturation
3. add patient sample or control serum containing antibodies (IgG)
patient serum = all antibodies in blood; if infected with HIV, IgGs to HIV will bind HIV antigen bound to dish
4. wash off unbound antibodies

3. 5. add 2° antibody that is conjugated
5. add 2° antibody that is conjugated (on Fc) to horseradish peroxidase enzyme
2° antibody = animal IgG antibody against Fc region of human of IgG (binds human IgGs that are bound to their antigens)
6. wash off unbound antibodies
-if no patient antibody bound to HIV, no 2°antibody will bind, enzyme will not be present
7. add substrates: Aminosalicylate and H2O2
-peroxidase reduces H2O2  O2 + H2O using salicylate as electron donor
-oxidized salicylate = brown

4. HIV positive = brown well: -patient IgG against HIV bind
-2°antibody against human IgG binds (has peroxidase linked to it)
-oxidation of salicylate  brown
(intensity indicates HIV antibody levels)
HIV negative = clear well
-no patient IgG against HIV
-no binding of 2° antibody
-peroxidase not present
-no reaction: substrates remain colorless
Edvo-Kit#271 page 7

5. Figure 74.1

6. - +
DS1
DS2

7. Exercise 72: Slide Agglutination (Indirect/Latex) Test
-beads have antibodies against coagulase and protein A
Figure 72.3
-only Staphylococcus aureus has coagulase and protein A
-antibodies + antigens = agglutination (visible clumps)

8. EDVOKIT#271: Simulation of HIV-1 Detection: ELISA
Lab 24 Goals and Objectives:
EDVOKIT#271: Simulation of HIV-1 Detection: ELISA
and Exercise 74: Enzyme Linked Immunosorbent Assay
Work in pairs: ten groups possible
Perform as indicated in supplement pg
Each group will need:
Micropipettor ( ) and tips
5 transfer pipettes
Beaker for waste solution and tips
Microtiter plate
Reagents: PBS, +, DS1, DS2
2°Ab (Secondary antibody: rabbit α human) Substrate: will be provided at time it is needed
Exercise 72: Slide Agglutination (Indirect/Latex) Test
Perform yourself at front of room or observe demo