1. Absorbance: Enzyme Linked Immunosorbent Assays (ELISA)

2. ELISA assays
Why? - Applications
How? – Principle
Example in KC4
What do you need to make this work?

3. Based on the VERY specific Antibody – Antigen bond
ELISA assays: why?
Antibody
Antigen
Based on the VERY specific Antibody – Antigen bond
Allows to identify a specific molecule in a complex biological mix (general purpose ELISA, used a lot in research labs).
Clinical application: detect specific antibodies in a blood sample (which contains a LOT of other molecules).

4. ELISA assays: why?
Example of applications:
Diagnostic of infectious diseases (Hepatitis, HIV, Rubella, Toxoplasmosis,…)
Quantification of hormones in blood (Progesterone, Corticosterone,…)
Follow-up of tumor marker concentrations to assess efficiency of cancer treatment
Quantification of all sorts of bio-molecules in complex biological samples.

5. ELISA assays
Why? - Applications
How? – Principle
Example in KC4
What do you need to make this work?

6. Samples dispensed and incubated
ELISA assays: how?
Example of a diagnostic kit
Samples dispensed and incubated
Antibody found in blood if the person is infected
Specific protein (antigen) coming from a specific micro-organism
Wells coated with antigens provided with the kit (clinical), or done in house (research)
Washing step 1
Positive sample
Negative sample

7. ELISA assays: how? Positive sample Negative sample
“Conjugate” added and incubated. Designed to recognize bound molecules.
Washing step 2
Positive sample
Negative sample

8. ELISA assays: how? Positive sample Negative sample
Substrate added and incubated. Reacts with the enzyme attached to the conjugate.
Reading step. Typically at 405nm, 450 nm or 490 nm (substrate dependent).
Positive sample
Negative sample

9. ELISA assays
Why? - Applications
How? – Principle
Example in KC4
What do you need to make this work?

10. ELISA assays: example in KC4
Quantitative ELISA assays:
Standard samples with known concentrations are used
Allows to generate a standard curve
Unknown sample concentrations are calculated from the curve
The final results is a concentration
Qualitative ELISA assays:
Control samples are used (positive, negative, cut-off…)
Allows to calculate one or several cut-off values
Unknown sample OD’s are compared to the cut-off(s)
The final results is an identifier (positive, negative, equivocal…)

11. ELISA assays: example in KC4
Quantitative ELISA assays

12. ELISA assays: quantitative assays
Reading parameters:
Typically end-point
Absorbance
Typically 405, 450 or 490 nm

13. ELISA assays: quantitative assays
Plate Layout:
Well Type: select “Standard”
Click on “…” to define the concentrations of the standards
Click “OK” and define the position of each standard on the plate map
Add “Blank” wells if any
Add “Assay Control” wells if any (positive control, negative control…)
Add sample wells

14. ELISA assays: quantitative assays
Typical plate layout:
6 standards in duplicate
Samples in duplicate
2 blank wells

15. ELISA assays: quantitative assays
Standard curve:
Go to “Curve” in KC4
Select the appropriate curve fit (see kit insert). If information not available, try “Linear Regression” and see results

16. ELISA assays: quantitative assays
Results:
KC4 automatically generates a standard curve
The concentrations of the unknown samples are automatically calculated from the curve.

17. ELISA assays: example in KC4
Qualitative ELISA assays

18. ELISA assays: qualitative assays
Reading parameters:
Same as quantitative assays:
Typically end-point
Absorbance
Typically 405, 450 or 490 nm

19. ELISA assays: qualitative assays
Plate Layout:
Well Type: select “Assay Control”
Click on “…” to define the type of control (e.g. POS, NEG…)
Click “OK” and define the position of each control on the plate map
Add “Blank” wells if any
Add sample wells

20. ELISA assays: qualitative assays
Typical plate layout:
Negative control in duplicate
Positive control in duplicate
Blank in duplicate
Sample wells in duplicate

21. ELISA assays: qualitative assays
Result interpretation
Go to “Cut-offs” in KC4
Enter cut-off(s) formula according to kit insert (e.g. mean OD of negative controls )
Enter the “Symbols” to be affected to the unknown samples after interpretation.

22. ELISA assays: qualitative assays
Results:
KC4 calculates the cut-off(s)
Samples are automatically interpreted

23. ELISA assays
Why? - Applications
How? – Principle
Example in KC4
What do you need to make this work?

24. ELISA assays: tips
Get a copy of the kit insert or the procedure before the demo. ELISA data analysis can be tricky, may require some preparation.
Ask in advance about the wavelength. Make sure you have the right filters if you demonstrate a filter-based reader.
Have an Absorbance Test Plate with you to prove the proper functioning of the reader if required.