General ELISA Procedure - Dako

Comments on Procedural Steps
Step 1 Coating of Wells with Antibody
Step 2 Washing
a. Use the immunoglobulin fraction of the antiserum for coating, not whole, unprocessed
antiserum.
b. The protein concentration of diluted antibody should be about 10 mg per L (optimal
concentration).
c. Coating at room temperature for 1 hour is possible, but coating at 4 °C overnight increases
sensitivity and precision of the assay.
d. Dilution of the antibody in buffer A gives ELISA results comparable to those obtained with the
commonly used 0.05 M carbonate buffer, pH 9.6.
e. Microwell plates may be precoated and stored at 4 °C for several months, if the coating is
performed as follows: Dilute the antibody in buffer A and make the solution 0.01% in
thimerosal. Add 100 µL antibody to each well and store the plate wrapped in plastic film at
4 °C.
The 0.1% Tween 20 in the washing buffer reduces the background and should be preferred
for 0.05% Tween 20, which is commonly used. An automated microplate washer is
recommended, in particular for ELISAs where infectious materials are handled and wastage
must be collected for disinfection.
Step 3 Incubation with Test Samples
a. Generally, biological samples should be diluted at least 1+1 to reduce the risk of non-specific
reactions.
b. Detection limit: Using the given general procedure, the detection limit is about 1 to 5 µg of
antigen per L of test sample. The sensitivity of a given antigen/antibody system might be
improved if the sample is made 3% in polyethyleneglycol MW 6000. Further, the sensitivity
will be improved by prolonging the incubation time, for example by incubating the plate -
covered with plastic film - at 4 °C overnight.
Step 5 Incubation with Peroxidase-Conjugated Antibody
a. It might speed up the reaction and improve the sensitivity if the peroxidase-conjugated
antibody is made 3% in polyethyleneglycol MW 6000. In addition, the sensitivity might be
improved if the incubation time is prolonged 1 or 2 hours.
b. The dilution of the peroxidase-conjugated antibody should be adjusted according to the
wanted measuring range. Usually, dilutions between 1:500 and 1:2000 are suitable.
Reagents
A. Coating Buffer 0.01 M Phosphate Buffer, 0.15 M NaCl, pH 7.2
NaH 2 PO 4 , H 2 O 0.35 g
Na 2 HPO 4 , 2H 2 O 1.34 g
NaCl 8.47 g
H 2 O ad 1 litre - check pH (7.2 ± 0.2)
Store at 4 °C. Stable for 2 months. (12 mL of buffer A is needed for one microwell plate).
B. Washing Buffer, 0.01 M Phosphate Buffer, 0.50 M NaCl, 0.1% Tween 20, pH 7.2
Dilution Buffer
NaH 2 PO 4 , H 2 O 0.35 g
Na 2 HPO 4 , 2H 2 O 1.34 g
NaCl 29.22 g
Tween 20
(Merck, No. 822184) 1 mL
H 2 O ad 1 litre - check pH (7.2 ± 0.2)
Store at 4 °C. Stable for 2 months. (1 to 3 litres of buffer B is needed for one microwell plate).
C. Chromogenic Buffer-containing
Substrate OPD tablets of 2 mg 4 (DAKO OPD Tablets, Code No. S 2045)
H 2 O 12 mL
Allow the tablets to dissolve, then add
30% H 2 O 2 5 µL
Prepare the reagent shortly before use. Remember that the chromogenic substrate should
have reached room temperature before use. After the addition of H 2 O 2 the reagent should be
watched a few minutes to ascertain that it remains slightly yellow. If the reagent colours
increasingly, it has been contaminated with peroxidase from unclean glassware or other
source.
The solution is stable at 24 °C for at least 2 hours if protected from light. The given volume is
suitable for one microwell plate.
Note: If only OPD tablets without buffer salts are available, the H 2 O should be replaced by a
0.1 M citric acid-phosphate buffer, pH 5.2, prepared as follows: citric acid,H 2 O 7.30 g,
Na 2 HPO 4 ,2H 2 O 13.71 g, H 2 O ad 1 litre. Store at 4 °C. Stable for 2 months.
D. 0.5 M Sulphuric Acid 28 mL 95 - 97% H 2 SO 4 is mixed into about
900 mL distilled water.
Note: Do not mix the water into the concentrated acid (risk of boiling).
Adjust the volume to 1 litre with distilled water. (About 15 mL of reagent D is needed for one
microwell plate).