ELISA

1. 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)

3. Capture antigen Capture antibody

5. http://webphysics.iupui.edu/webscience/bio_archive/goodfor3.htmlhttp://webphysics.iupui.edu/webscience/bio_archive/goodfor3.html • http://www.whfreeman.com/kuby/content/anm/kb07an01.htm

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

7. 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

8. 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

9. 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

10. 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.

11. 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.

12. 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

13. 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

14. 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

15. 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?

16. 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.

18. Western Blot Used to confirm an ELISA positive for HIV

19. Western blot

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

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

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

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

24. 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.

25. 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.

26. 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.