Absorbance Enzyme Linked Immunosorbent Assays ELISA ELISA assays

Absorbance: Enzyme Linked Immunosorbent Assays (ELISA)

ELISA assays • • Why? - Applications How? – Principle Example in KC 4 What do you need to make this work?

ELISA assays: why? Antigen Antibody • 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).

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.

ELISA assays • • Why? - Applications How? – Principle Example in KC 4 What do you need to make this work?

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

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

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

ELISA assays • • Why? - Applications How? – Principle Example in KC 4 What do you need to make this work?

ELISA assays: example in KC 4 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…)

ELISA assays: example in KC 4 Quantitative ELISA assays

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

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

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

ELISA assays: quantitative assays Standard curve: • Go to “Curve” in KC 4 • Select the appropriate curve fit (see kit insert). If information not available, try “Linear Regression” and see results

ELISA assays: quantitative assays Results: • KC 4 automatically generates a standard curve • The concentrations of the unknown samples are automatically calculated from the curve.

ELISA assays: example in KC 4 Qualitative ELISA assays

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

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

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

ELISA assays: qualitative assays Result interpretation • Go to “Cut-offs” in KC 4 • Enter cut-off(s) formula according to kit insert (e. g. mean OD of negative controls + 0. 200) • Enter the “Symbols” to be affected to the unknown samples after interpretation.

ELISA assays: qualitative assays Results: • KC 4 calculates the cut-off(s) • Samples are automatically interpreted

ELISA assays • • Why? - Applications How? – Principle Example in KC 4 What do you need to make this work?

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.