1. Enzyme-linked Immunosorbent Assay
ELISA
Enzyme-linked Immunosorbent Assay

2. 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)

6. 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

7. 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

8. 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.

9. 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

10. 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.

11. 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

12. 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.

13. 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

14. 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?

15. 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.

16. 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.

17. 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.

18. 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.

19. Western blot

20. Indirect Western immunoblot for HIV diagnosis

21. Indirect Western immunoblot for HIV diagnosis

22. Two or more bands are required to be considered positive.