Colorimetric Assays

Enzyme-linked Immunosorbent Assay (ELISA)

Colorimetric assay used to quantify protein expression through cell lysates or cell secretions.

Sandwich ELISA steps

1) Bind capture protein to empty dish/well.

2) Block

3) Add sample

4) Wash *

5) Add primary antibody (same as capture)

6) Wash *

7) Add secondary with enzyme

8) Wash *

9) Add substrate

Multichannel pipette

Used to time-normalize samples of the assay. Typically an 8 or 12 channel is used.

Biotek or Tecan

Brand of microplate readers that utilize UV Vis spectrophotometer. Contains software in which sample info can be entered.

Eliza kits

Kits of target protein for ELISA. Contains antibodies, enzymes, and protein standard.

Protein standard

Given amount of protein in ELISA kit used to generate a standard curve (dilutions have to be produced).

Commonality of Assays

1) Colorimetric

2) Luminescence

3) Fluorescence

ISO10993

Bioevaluation of medical devices to ensure materials does not leech and is not cytotoxic.

Live-Cell Protease Viability Assay (CellTiter-Fluor Cell Viability Assay)

Cell-permeable fluorogenic protease substrate enters the cell and gets cleaved by protease, generating a fluorescent signal that is proportional to viable cells.

CytoTox 96 Non-Radioactive Cytotoxicity Assay

Detects conversion of a tetrazolium salt (INT) into a red formazan product, measured by color absorbance.