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 117-127 Each group will need: Micropipettor (100- 1000) 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

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

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

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

Figure 74.1

- + DS1 DS2

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)

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 117-127 Each group will need: Micropipettor (100- 1000) 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