ELISA
1.1.5
Pathogen
• Greek: “Path” = disease or
suffering, “gen” = producer or
beginning
• A microorganism that causes
disease.
• Pathogens have DNA, so
determining their nucleotide
sequences can be used to
specifically identify them.
The Immune System
When you get sick:
• Your B lymphocytes make antibodies to attack antigen on the surface
of the pathogen
Antigen
• Protein or other macromolecule on
the surface of a cell.
• All cells have hundreds of different
antigens on their surfaces, and they
have many functions. One is
self/non-self recognition.
• Self-antigen – recognized by your
WBCs as nonthreatening. Self-
antigen is not supposed to provoke
an immune response.
• Non-self antigen – recognized by
WBCs as foreign, and causes them
to launch an immune response.
• Interacts with receptors on other
cells (like leukocytes), or with
antibodies.
Antibody
• Quaternary proteins that have a Y-
shape.
• 2 heavy chains
• 2 light chains
• Tips of the Y have a unique shape
that fits with one particular antigen.
• Produced by lymphocytes when
antigen is present.
• Can be released into the blood
• Can be embedded in WBC cell
membrane
Antigen-Antibody Interaction
ELISA
Enzyme Linked Immunosorbent Assay
ELISA Principles
• Determine presence of a
substance
• Antigen (Indirect ELISA)
• Antibody (Sandwich ELISA)
• Common health diagnostic
tool
• Performed on ELISA plates
made up of multiple wells.
ELISA Principles
Based on antigen/antibody interactions
Types of ELISA
Types of ELISA
Direct ELISA Indirect ELISA
• Tests for presence of antigen in • Tests for the presence of
the patient sample. antibody in the patient sample.
• Useful early in infection, before • Useful in detecting infection in
antibody production begins. later stages, after antibody
• Uses: Testing for dangerous, fast production has started.
acting pathogens such as • Uses: Test for successful
meningitis. vaccination, chronic viral
infections such as HIV.
• Antigen: Sample of patient serum is
introduced. If correct antigen is present, it will
The major steps: stick to the inside of the well. (This doesn’t
keep other antigen from sticking, but the test
will only show results for antigen of interest.)
• Primary antibody: Antibody specific for the
antigen is added. If the antigen is in the well,
it will bind with the antibody.
• Secondary antibody: Specific for the primary
antibody. It has an extra enzyme molecule
linked to it that will cause a color change in
the next step. Again, none of this will happen
if the antigen was not present in the patient
sample to begin with!
• TMB substrate: Specific for the enzyme. If the
enzyme is a part of this “sandwich” we have
been building, a color change will occur.
Qualitative Results
• Evaluate a non-numerical quality
of experimental results.
• In this case, the intensity of the
color change.
• Deeper color indicates more
antigen was present in the
patient sample.
• Lighter color, there was little
antigen.
Standard Curve
• Samples of known antigen (or antibody) concentration prepared using
serial dilution
• Serial dilution – dilutes the sample by specific amounts over several wells. The
concentration of the sample can be calculated by fractions
• The color of the unknown sample is compared to the known
concentration of one of the wells of the standard curve
Patient Sample
Standard Curve
Quantitative Results
• Numerical data. Considered more accurate,
and more desirable for reporting results.
• Spectrophotometer - Measures
concentration of your sample by measuring
the amount of light the sample absorbs
(absorbance)
• The spectrometer shoots a beam of light
through the sample
• The color of the substrate in the sample
blocks some of the light.
• A detector records how much light of that
color makes it through.
False Positive ELISA
• Sometimes ELISA comes back positive for a particular antigen when
the individual is actually clear.
• Why? Reasons involve sensitivity to a cell membrane protein called
Human Leukocyte Antigen (HLA). Pathogens can incorporate HLA
from its host into themselves. If a patient has been exposed to HLAs
from other people, they may test positive when they are not.
• Frequent blood transfusions ~ HLAs from the donor are in the patient’s
system.
• Multiple pregnancies ~ HLAs from the children are in the mother’s system.
• Exposure to animals
Strep Test:
HIV Test:
1.1.5 Results
100
μg/mL
ELISA Key Stop
(EDVOTEK)
Primary
TMB antibody
• A - Sue
• B - Jill
• C - Maria
+ Secondary
• D - Marco Control
ELISA antibody
• E - Maggie
• F - Anthony
• G - Arnie - Dilution
• H - Wanda Control
buffer
• I - Alvin Antigen
A B C D
