Enzyme-linked Immunosorbent Assay 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)

Steps for our HIV Indirect ELISA Addition of antigen Wash (Block available well sites) Add controls and unknowns; incubate Add enzyme-linked secondary antibody; incubate What does this antibody recognize? What is the primary antibody? 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 antibodies with antigens in the well Sensitivity See next slide Controls Known positive Known negative

What contributes to the specificity of the assay? Antigens used – when coating the plate the antigen need not be purified, but should comprise at least 3% of the protein in the coating solution Antigen/antibodies used – Direct – Capture antibodies should be of high affinity and high specificity and should be screened for unwanted cross-reactions Indirect – 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.

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 antibodies-amplification Affinity of the antigen/antibody interaction

Additional contribution to sensitivity for ELISAs Direct Use of polyclonal antibodies- more than one antibody can capture antigen. 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.

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 – Is specific antigen or antibody present in the sample being analyzed? Quantitative – If specific antigen or antibody is present in the sample being analyzed, how much is present?

How can ELISAs be used for quantitative determinations of Ag. or Ab How can ELISAs be used for quantitative determinations of Ag. or Ab. concentrations? Keep all experimental conditions constant between experiments. Include a standard curve on each plate. Analyze all samples in at least duplicate. 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 constant sensitivity specificity 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 constant sensitivity specificity 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.

Western blot

Indirect Western immunoblot for HIV diagnosis

Indirect Western immunoblot for HIV diagnosis

Two or more bands are required to be considered positive.