Download Secondary antibody

Ex. 28: 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
• Microplate strips are made of
polystyrene
• Hydrophobic side chains in
amino acids bind to the
polystyrene wells
WASH
• Remove the liquid from the sample
wells by tipping the microplate strip
upside down and discarding the
solution into a beaker.
• Firmly tap the strip a few times
upside down onto a paper towel.
• Discard the paper towel.
• Using a disposable transfer pipette
almost fill the wells with wash buffer.
• Remove the wash buffer following
the procedure above. Always discard
the used paper towels
• Repeat the 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
• Wash buffer contains phosphate
buffered saline (PBS) to keep
ABs in stable environment that
helps keep their structure
• Also contains Tween 20: a
nonionic detergent that helps to
remove non-specifically 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 this is happening?
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. Rinse
ELISA Procedures
Summary
Add serum antibodies
(patient samples) to the
appropriate wells. Incubate
for 5 min. Rinse
Add the enzyme-linked
antibody to all wells.
Incubate for 5 min. Rinse
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