Ex. 27: HIV ELISA,
AIDS Diagnostic Tool
Human
Immunodeficiency
Virus (HIV)
• First diagnosed in 1981
• Over 20 million deaths worldwide,
over a half million in the United
States
• Over 40 million currently infected,
over a million in the United States
• Education effective in limiting the
spread of HIV/AIDS
ELISA
Antibody Structure Review
Enzyme-Linked
Immunosorbant
Assay
ELISA tests are
based on
antibody
molecules.
Heavy
chain
Disulfide
bonds
Light
chain
Antigens
HIV ELISA is an Indirect ELISA
ELISA-HIV
Test
Detecting
Antibodies in
Serum
•After 4-8 weeks of exposure to
the HIV virus: body will have
produced detectable level of
antibodies against HIV
•HIV-ELISA detects presence
of serum antibodies against
HIV protein antigens
Step One
Label wells
and add
antigen
Label the 12-well strip:
– First 3 wells: positive controls “+”
– Next 3 wells: negative controls “-”
– Remaining wells patient samples
(3 wells for each patient)
Transfer 50µl of purified antigen
(AG) into all 12 wells
Wait 5 minutes for the antigen to
bind
Microplate Strips / Microtiter Plates
• are made of polystyrene
• Hydrophobic side chains in
amino acids bind to the
polystyrene wells
Most
important
step:
WASH
1. Remove liquid from sample wells
by tipping microplate strip upside
down and discarding solution into
big glass petri dish
2. Firmly tap strip a few times upside
down onto a paper towel
3. Discard paper towel
4. Using disposable transfer pipette
almost fill wells with wash buffer
5. Remove wash buffer following
procedure above. Always discard
the used paper towels
6. Repeat wash step
Step Two
Add controls
and patient
samples
• Add 50 µl of positive control to
1st three wells
• Add 50 µl of negative control to
2nd three wells
• Add 50 µl of patient sample A to
3rd set of three wells
• Add 50 µl of patient sample B to
last 3 wells
• Incubate at room temperature
for 5 minutes.
• Wash twice
Wash
Buffer
contains …
• Phosphate buffered saline
(PBS) to keep abs in stable
environment that helps keep
their structure
• Tween 20: Nonionic detergent
that helps to remove nonspecifically bound proteins
(reduces background)
Step Three
• Add 50 µl of the enzyme-linked
secondary antibody to each well
Add enzymelinked AB
• Incubate at RT for 5 minutes.
• 2° ab (enzyme-linked antibody)
will only bind to primary ab
(serum antibody)
• 2° ab specifically
recognizes constant
region of 1° ab
• In which wells do you
predict binding will occur?
Step Four
Add enzyme
substrate
• Wash the enzyme-linked
secondary antibody from
polystyrene wells as before
• WASH 3X
• Add 50µl of the enzyme
substrate to each well
• Incubate at room temperature
for 5 minutes
• Positive samples will begin to
turn blue
ELISA ANIMATION
And . . . .
one more ELISA animation
Add purified ag to all the wells.
Incubate for 5 min. Wash.
ELISA Procedures
Summary
Add serum antibodies
(patient samples) to the
appropriate wells. Incubate
for 5 min. Wash.
Add the enzyme-linked
antibody to all wells.
Incubate for 5 min. Wash.
Add enzyme substrate
to all wells. Incubate
for 5 min.
Reagents Summary
1. Purified HIV Antigen
2. Primary antibody (Patient serum samples)
3. Secondary antibody: conjugated polyclonal
anti-human antibodies made by goats.
Conjugate is horseradish peroxidase (HRP)
4. Substrate for HRP: 3,3’,5,5’ –
tetramethylbenzidine (TMB) – a colorless
solution that turns blue when oxidized by
HRP
ELISA Kit
Results
Clear
Determination
Of Positive
And Negative
Results