uncoated ELISA

I. Required material

Reagents
- A primary antibody capture, specific to the target molecule
- A second primary antibody detection, specific to the target molecule
- A recombinante molecule to use as a standard
- Detection Solutions: e.g. Streptavidin-HRP and TMB

Buffers
- Coating Buffer: e.g. PBS
- Blocking Buffer: e.g. PBS with 5%BSA
- Wash Buffer: e.g. PBS with 0.05%Tween
- Diluent for Sample and Standard: e.g. PBS 1%BSA
- Stop Solution: e.g. diluted H2SO4

Material - 96 well microtitre plates
- Plate covers/sealers
- 5 ml and 10 ml graduated pipettes
- 20 µl to 1,000 µl adjustable single channel micropipettes with disposable tips
- 50 µl to 300 µl adjustable multichannel micropipette with disposable tips
- Multichannel micropipette reservoir
- Beakers, flasks, cylinders necessary for preparation of reagents
- Device for delivery of wash solution (multichannel wash bottle or automatic wash system)
- Microtitre plate reader capable of reading at 450 nm



II. Experimental period
- 1 to 2h of labelling
- 3h of procedure
- 30 min of various washes
- 10 min of reading
- TOTAL : 4 to 5 hours


III. Procedure
Coating - Dilute the capture antibody to the appropriate concentration (as per insert) using coating buffer (10ml final volume required per plate)
- Add 100 µl of the capture antibody solution to all wells of the plate, cover and incubate overnight at 4°C
- Aspirate the contents of each well, then wash the plate at least twice using 400 µl of wash buffer in each well.

Note : Following the final wash ensure all remaining buffer is removed by gentle blotting on absorbent paper

- Add 250 µl of blocking solution to all wells of the plate, cover and incubate at RT for 2 hours
- Aspirate the contents of each well, then wash the plate at least twice using 400 µl of wash buffer in each well, the plate is now ready to run the assay
Assay procedure
- Add 100 µl of prepared standard, sample and control dilutions to appropriate wells, cover the plate and incubate at RT for a minimum of 1 hour
- Aspirate the contents of each well, then wash the plate at least twice using 400 µl of wash buffer in each well
- Add 100 µl of the diluted detection antibody (diluted as per insert) to every well, cover the plate and incubate at RT for a minimum of 1 hour
- Aspirate the contents of each well, then wash the plate at least twice using 400 µl of wash buffer in each well
- Add 100 µl of the Streptavidin-HRP solution to all wells, cover the plate and incubate at RT for 30mins
- Aspirate the contents of each well, then wash the plate at least twice using 400 µl of wash buffer in each well
- Add 100 µl of the TMB solution to all wells, cover the plate and incubate at RT for a minimum of 10mins
Note : Incubation time of the substrate solution is usually determined by the ELISA reader performance. Many ELISA readers only record absorbance up to 2.0 O.D. Therefore the colour development within individual microwells must be observed by the analyst, and the substrate reaction stopped before positive wells are no longer within recordable range
- Add 100 µl of stop solution to all wells and within 30mins measure the absorbance readings at 450nm (when available with 620nm as the reference wavelength, 610-650 is acceptable)