T5 | Elisa & Other Colorimetric Assays

What does Elisa quantify?

Protein expression/secretion

What samples do you run Elisa from

In vitro lysates or protein in a serum from blood draws

How is Elisa different from other colorimetric assays?

It requires antibodies

Draw antibodies for Elisa to detect IL-10 in mouse blood

What are the general steps for Elisa

1. Grow fibroblasts

2. Does fibroblasts with X

3. Add sample to antibody, add another antibody, add enzymatic stop

4. Read the absorbance on blank, calibrator, and Elisa plate X min after enzymatic stop

5. Create a standard curve from the diluted calibrator

6. Plug in Elisa ABS into curve to get []

Describe how Elisa results would look

More protein = darker color = high absorbance = high concentration

What does Elisa stand for

enzyme linked immunosorbent assay

What is the ISO for cytotoxicity testong

0.5-6.0 cm²/mL media for 72 hours @ 37C

What can you measure with cytotoxicity

If the cell membrane is intact. Live/dead

What does XTT assay quantify, what color is it

• if the mitochondria/ATP is working. Quantifies ATP

• Yellow → Orange is good

What does Calcein AM + Ethidi homofimer-1 quantify and what color is it

Live cells = green

Dead = red

Quantified membrane integrity

What does Annexin V quantify and what color is it

• early apoptosis

• Early apoptosis = green, late/dead =green+red

What is the N for these assays

6 wells per sample

What are the general colorimetric assays steps

1. Seed the same #cells/well

2. Grow cells for X days

3. Treat/apply variable