Enzyme Linked Immunosorbent Assay
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Transcript of Enzyme Linked Immunosorbent Assay
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.