Absorbance: Enzyme Linked Immunosorbent Assays (ELISA)

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

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).

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 KC4 What do you need to make this work?

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

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

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

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

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…)

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

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

ELISA assays: example in KC4 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 KC4 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: KC4 calculates the cut-off(s) Samples are automatically interpreted

ELISA assays Why? - Applications How? – Principle Example in KC4 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.