Lab 24 Goals and Objectives: EDVOKIT#271: Simulation of HIV-1 Detection: ELISA Work in pairs: ten groups possible Perform as indicated (supplemental packet.

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Lab 24 Goals and Objectives: EDVOKIT#271: Simulation of HIV-1 Detection: ELISA Work in pairs: ten groups possible Perform as indicated (supplemental packet with corrections) Exercise 74: Slide Agglutination (Indirect/Latex) Test Perform yourself at front of room or observe demo

HIV virus Lentivirus - a member of the retrovirus family (RNA) Causes acquired immunodeficiency syndrome (AIDS) HIV infects vital cells in the human immune system Helper T cells (specifically CD4 + T cells) Macrophages Dendritic cells HIV testing is initially by an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV.

Edvo-Kit#271 page 7

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 2º antibody that is conjugated (on Fc) to horseradish peroxidase enzyme 2º 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 2º antibody will bind, enzyme will not be present 7. Add substrates: Aminosalicylate + H 2 O 2 Horsredish peroxidase H 2 O 2 O 2 + H 2 O salicylate as electron donor oxidized salicylate = brown

- + DS1 DS2

Exercise 74: Slide Agglutination (Indirect/Latex) Test -beads have antibodies against coagulase and protein A -only Staphylococcus aureus has coagulase and protein A -antibodies + antigens = agglutination (visible clumps)

Lab 24 Goals and Objectives: EDVOKIT#271: Simulation of HIV-1 Detection: ELISA Work in pairs: ten groups possible Perform as indicated (supplemental packet with corrections) 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 74: Slide Agglutination (Indirect/Latex) Test Perform yourself at front of room or observe demo