ELISA (Enzyme-Linked ImmunoadSorbent Assay)

A. You will need the following solutions

PBS (Phosphate-Buffered Saline, Ca/Mg free) :

Solute	1x	10x
NaCl	8g	80g
KCl	0.2g	2g
Na2PO4 (anhydrous)	1.15g	11.5g
KH2PO4 (anhydrous)	0.2g	2g

N.B.: Na2HPO4.2H2O can also be used -> add 1.42g for 1x and 14.2g for 10x PBS.

Make up to 1 litre with dd H2O.

PBS wash:

100ml 1x PBS

10ul Tween (0.01%, final concentration)

Citrate/Acetate Buffer pH6.0:

1. 500ml 0.1M Sodium Acetate

Sodium acetate - 4.1g.

dd H2O - 500ml.

2. 100ml 0.1M Citric acid

Citric acid - 2.1g.

dd H2O - 100ml.

Titrate 1 with 2 to a final pH of 6.0. Citrate/Acetate Buffer, pH6 can be stored frozen at -20°C.

TMB Solution - 10mg/ml in DMSO (store at 4°C).

B. The following details the method for use with TMB as the substrate (cover wells with clingfilm during incubations to prevent evaporation).

1) Add 3ul H2O2 to 20ml of Citrate/Acetate buffer. To every 5ml of buffer add 50ul TMB solution.

N.B.: Colour change is from colourless to blue (yellow when acid is added).

2) Coat plate with antigen stock solution (1ug/ml in PBS, 50ul/well) for at least 24hrs or over the weekend at 4°C or O/N at 37°C.

3) Remove antigen and wash 3x with 1x PBS.

4) Block with 1% BSA (100ul/well) in 1x PBS for at least 1hr at 37°C.

5) Remove BSA by flicking plate.

6) Wash 3x with 1x PBS containing 0.01% tween

7) Add anti-serum (diluted as appropiate, 1/100 etc. with 1x PBS) and incubate for 2hrs @ 37°C.

N.B.: Use positive and negative controls.

8) Wash 4x with 1x PBS containing 0.01% tween.

9) Add peroxidase conjugate (50ul/well) diluted 1:1000 with 1% BSA (diluted in washing solution as in 8)) and incubate for 1hr @ 37°C.

N.B.: Use PRAM (Peroxidase-conjugated Rabbit Anti-Mouse) for Ab raised in mice and PSAR (Peroxidase-conjugated Swine Anti Rabbit) for Ab raised in rabbits.

10) Wash 4x with 1x PBS containing 0.01% tween.

11) Add substrate (100ul/well).

12) Incubate for 1-5 mins in the dark at RT. Check reaction for colour change. Wells should turn blue.

13) Stop reaction by the addition of 25ul 0.1M H2SO4/well.

14) Read OD @ 450nm for TMB.