Enzyme Linked Immunosorbent Assay

ELISADetection 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

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

ELIS

A

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 10-15 min. at 37C

NOT IN A WATER BATH!!

C.

Remove solutions from each well

Rinse with PBS

D.

Add .1 ml of labeled 2nd Ab to each well

Remove 2nd 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.