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