ELISA

Enzyme-linked Immunosorbent Assay

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)

Capture antigenCapture antibody

http://webphysics.iupui.edu/webscience/bio_archive/goodfor3.html

http://www.whfreeman.com/kuby/content/anm/kb07an01.htm

Antibody against Human IgG, raised in rabbit.(Rabbit was injected with human IgG antibodies)

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.

Western Blot

Used to confirm an ELISA positive for HIV

Western blot

Indirect Western immunoblot for HIV diagnosis:how the test strips are made

Indirect Western immunoblot for HIV diagnosis:how the test is perfomed

Two or more bands are required to be considered positive.

The next 3 slides Are for your own information Will not be covered on the exam

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.

Sample criss-cross results for a Direct ELISA with Capture Antibody

constantspecificitysensitivi

ty

The investigators would need to double check that under the conditions they choose based on the criss-cross, the minimum positive is within the dynamic range of the standard curve.