ELISA

Definition(Wikipedia

Enzyme-linked immunosorbent assay (ELISA), is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample.

Buffers and materials

Using 96 well plates for RIA/EIA (HIGH BINDING) NUNC company

Coating buffer: 0.1 M NaHCo3 pH 9.3 (adjust with 1 M NaOH)

Washing buffer (PBS-T): 1x PBS + 0.05 % (v/v) Tween 20

Blocking buffer: Dissolve casein (purified powder) to 2 g/l in PBS-T. Heat up to 37°C and stir until casein is dissolved (takes some time!)

Protocol

- Shake the plate while incubating at RT. - Dilute antibodies in blocking buffer!

1) Coat the plate o Use antigen with a concentration of 5 g/ml in coating buffer o Add 50 l/well o Incubate o/n @ 4°C

2) Wash the plate 3 times with washing buffer (using plate washer) with 200µl/well

3) Block for 1 h @ RT with blocking buffer (200 l/well)

4) Wash the plate 3 times with washing buffer (using plate washer)

5) Add 50 l/well of primary antibody (1:1000, 1:5000 dilution of serum in blocking buffer), Incubate for 1 h @ RT

6) Wash the plate 5 times with washing buffer (using plate washer)

7) Add 2nd antibody (detection ab) in a dilution of 1:500 (HRP-goat anti-rabbit, 10 l ab + 5 ml blocking buffer) o Incubate for max. 1 h @ RT (here: 45 min)

8) Wash the plate 5 times with washing buffer (using plate washer)

9) Add 100 l of substrate (2,2`-Azino-bis(3-ethylbenzthiazoline-6-sulforic acid; ABST (NH4)2 Sigma A3217 (100 ml liquid); A1888 powder) o Measure absorbance @ 405 nm (or 620 nm) after color develops o Measure between 5 and 13 min (here: 17 min, Mike did a kinetics with HRP antibody, after 17min best result)

Software

Open the program (omega)  USER run  quick start  NUNC96, 405 nm, name the plate  measure Results  open last test run  save the data  transfer the data by USB stick