Lab 24 Goals and Objectives:

EDVOKIT#271: Simulation of HIV-1 Detection: ELISAWork in pairs: ten groups possiblePerform as indicated (supplemental packet with corrections)Each group will need:

Micropipettor (100- 1000) and tips5 transfer pipettesBeaker for waste solution and tipsMicrotiter plateReagents: 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) TestPerform 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 (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 + -peroxidase reduces H2O2 O2 + H2O using salicylate as electron donor-oxidized salicylate = brown

H2O2

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

-+DS1DS2

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: ELISAWork in pairs: ten groups possiblePerform as indicated (supplemental packet with corrections)Each group will need:

Micropipettor (100- 1000) and tips5 transfer pipettesBeaker for waste solution and tipsMicrotiter plateReagents: 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) TestPerform yourself at front of room or observe demo