ELISA
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Transcript of ELISA
ELISA Enzyme Linked Immunosorbent
Assay All ELISAs depend on an enzyme-
linked second antibody to produce visible signal
Direct assays Detect antigen in the sample (e.g., HIV viral
proteins) Indirect assays
Detect antibodies in the sample (e.g., antibodies against HIV proteins)
http://webphysics.iupui.edu/webscience/bio_archive/goodfor3.html
http://www.whfreeman.com/kuby/content/anm/kb07an01.htm
Steps for our HIV Indirect ELISA Addition of antigen Wash (Block available well sites) Add controls and unknowns (i.e., the sample); incubate Wash Add enzyme-linked secondary antibody; (first team to start
this incubation, let me know) incubate What does this antibody recognize? What is the primary antibody?
Wash Add substrate; incubate Analyze results
Indirect ELISA for HIV antibodies Sample – patient blood sample Probe – HIV antigens coating the well of the
plate Specificity
Specific interaction of patient’s antibodies with antigens in the well
Sensitivity See next slide
Controls Known positive Known negative
What contributes to the specificity of the assay?
Indirect assay – capture antigen Capture antigen should be of high affinity
for the target antibodies and should be screened for unwanted cross-reactions with antibodies from individuals who are negative for the condition of interest.
When coating the plate, the antigen need not be purified, but should comprise at least 3% of the protein in the coating solution
Direct assay – capture antibody Capture antibodies should be of high affinity
and high specificity and should be screened for unwanted cross-reactions
What contributes to the sensitivity of an ELISA assay? Blocking- raises signal to noise ratio Washing- raises signal to noise ratio Use of enzyme-conjugated secondary
antibodies amplification of visualization signal
Affinity of the antigen/antibody interaction The higher the affinity, the less sample is
needed.
Additional contribution to sensitivity for ELISAs Direct
Use of polyclonal antibodies different antibodies against more
than one antigenic determinant on the antigen increase chances of antigen capture.
Indirect Use of more than one appropriate
antigen will capture more antibodies from the sample.
What kinds of controls should be included in an ELISA? Positive control – known positive
tests that the reagents are all working properly
Negative control – known negative assures that there are no cross-
reactions between the reagents that lead to false positives
False Positives
What is a false positive? The results say that whatever is being assayed
for is present, even though it is not present. What can cause a false positive in an
ELISA? Failure to wash carefully “Cross-reactivity” of the antibodies involved
Example: Some antibodies against flu have given false positives for the HIV ELISA.
The antibodies against flu antigens “cross-react” against HIV antigens
Note: Cross-reacting antigens need only be sufficiently similar, not identical
Advantages of ELISAs over other assays for antigen or antibody
Sensitivity Long shelf-life of reagents Lack of radiation hazards Ease of preparation of reagents Speed and reproducibility of the
assays No sophisticated equipment is
required
What kind of information can be determined from an ELISA?
Qualitative – The assay detects presence of specific antigen or antibody in the sample being analyzed.
Quantitative – If specific antigen or antibody is present in the sample being analyzed, the assay detects how much is present.
Which is our version?
How can ELISAs be used for quantitative determinations of Ag. or Ab. concentrations?
Keep all experimental conditions constant between experiments.
Analyze all samples in at least duplicate.
Include a standard curve on each plate. The concentration of the reagent
being quantified must be in the dynamic range of the standard curve.
How do you determine the optimal concentrations of all reagents used in the system?
Perform a criss-cross serial dilution experiment in which one reactant is kept constant and the other two reactants are varied.
Test both homologous (the antigen of interest) and heterologous (completely unrelated) antigens.
Sample criss-cross results for a Direct ELISA with Capture Antibody
constantspecificitysensitivi
ty
The investigators wanted their instrument to read 1000 at the lowest level of antigen to be considered “positive” in a diagnostic situation (in this case, 0.78) and to read 10 or less for a negative control.