Enzyme Linked Immunosorbent Assay ELISA Detection of Anti-HIV (This outline summarizes key points of ELISA protocol as outlined in the handout.)

ANTI-HIV Simulate LABORATORY  Introduction to the protocol  Laboratory Steps

A diagnostic test for Antibodies to HIV Enzyme Linked Immunosorbent Assay (ELISA) Antibodies

Antigens Immune system Non-self

Objective: to use the ELISA to detect anti-HIV from 2 donors.

Supplies needed /group  4 transfer pipets  1 transfer  1 set of samples  1 microtiter plate (12 wells)  1 beaker of Phospate buffered saline (PBS)  1 empty beaker for “waste”

Transfer pipets  Try to approximately add the  Same volume to each well.  Approximate volume 0.l ml

Transfer Pipets for rinsing steps  Label transfer pipets PBS 1 PBS 2 PBS 3 PBS 4

Let’s ELISA

A.  Add.1 ml of antigen to each well  Remove antigen solution  Rinse well one time with PBS Incubate 5-10 minutes at room temperature

B. Add 0.1 ml of  PBS  + control  Donor 1  Donor 2  Row 1  Row 2  Row 3  Row 4 Row 1 Row 2 Row 3 Row 4 Incubate min. at 37C NOT IN A WATER BATH!!

C.  Remove solutions from each well  Rinse with PBS

D.  Add.1 ml of labeled 2 nd Ab to each well  Remove 2 nd antibody solution  Rinse wells with one time PBS Incubate5-10 minutes at 37C

E.  Add.1 ml of substrate to each well  Identify wells with anti-HIV 5-10 minutes at 37C

OVERVIEW

Microplate Readers

Examples of Antibody dilutions

Hand-In:  A drawing of the steps involved in the ELISA  The results of your laboratory results  The interpretation of your results  Provide an example of how you could obtain a false positive and a false negative result.