Equipment
Microwell Plate We recommend NUNC MaxiSorp, Code No. 439454, flat bottom polystyrene plate. This
plate contains 8 x 12 wells which hold 350 µL each, and it is well-suited for quantitative
ELISA. (NUNC, Kamstrup, DK-4000 Roskilde, Denmark).
Multipipette An 8-channel 100 µL pipette is a good help for even small-scale work.
Washing Device NUNC Immuno Wash, Code No. 470173 (8-channel), or Code No. 455492 (12-channel),
are manually operated washing devices. They may be of use particularly when there is a
risk that the samples tested in ELISA contain infectious material, and therefore must be
collected for subsequent disinfection. (NUNC, Kamstrup, DK-4000 Roskilde, Denmark).
Microplate Washer In our hands, Denley microplate washers are very efficient with unusually low carry-over
contamination. (Denley Instruments Ltd., Natts Lane, Billingshurst, West Sussex,
RH14 9EY, England).
General ELISA Procedure
Quantitation of Trace Amounts of Antigens by Double
Antibody Sandwich ELISA on Microwell Plates
1. Coating of Wells with Antibody
100 µL of antibody diluted in buffer A is added to each well. The antibody should be
directed against the antigen to be determined.
Cover the plate with plastic film or aluminium foil and incubate at 4 °C overnight.
2. Washing
Wash the wells 4 times with buffer B using a microplate washer. Alternatively, wash manually
3 times as follows:
Empty the plate by inversion over a sink. Tap the inverted plate against some layers of soft
paper tissue to remove residual liquid. Wash the plate by filling the wells by immersion in
buffer B. Leave on the table for 3 minutes. Empty the plate as described above and repeat
washing two more times.
3. Incubation with Test Samples
100 µL of test sample or standard diluted in buffer B is added per well.
Cover the plate and incubate at room temperature for 2 hours.
4. Wash as described in step 2.
5. Incubation with Peroxidase-Conjugated Antibody
100 µL of peroxidase-conjugated antibody diluted in buffer B is added to each well.
Cover the plate and incubate at room temperature for 1 hour.
The peroxidase-conjugated antibody should be directed against the antigen to be determined.
6. Wash as described in step 2. When washing manually it is mandatory at this step that
the washing buffer in the reservoir is totally exchanged after the first of the 3 washes. If this is
not done, a high background staining will occur.
7. Colour Development
100 µL of chromogenic substrate C is added to each well.
Cover the plate and incubate for 15 minutes, or until a suitable colour has developed. The
plate should preferably be protected against light during this incubation.
8. Stopping the Colour Development
Stop the reaction by adding 100 µL 0.5 M H2SO4 (reagent D) to each well.
It is important for quantitative measurements that each well has been incubated with colour
reagent for exactly the same length of time.
9. Reading of Results
Read results directly through the bottom of the microwell plate using an automated or semi-
automated photometer (ELISA-reader). Read the plate at 490 nm. The subtraction of the
absorbance at a reference wavelength (between 620 and 650 nm) is recommended, but not
essential.
Alternatively, read results within 3 hours in a photometer at 490 nm using a cuvette requiring
no more than a 200 µL volume. The cuvette can conveniently be emptied by a piece of plastic
tubing connected via a reservoir to a vacuum pump or to water suction.
10. Plot the standard curve on semilogarithmic paper with A490 nm as ordinate and log10 concentration of
standard as abscissa.
DAKO A/S • Produktionsvej 42 • DK-2600 Glostrup • Denmark • Tel. +45 44 85 95 00 • Fax +45 44 85 95 95 • www.dako.com
 Comments on Procedural Steps Reagents

A. Coating Buffer 0.01 M Phosphate Buffer, 0.15 M NaCl, pH 7.2

Step 1 Coating of Wells with Antibody
NaH2PO4, H2O 0.35 g
a. Use the immunoglobulin fraction of the antiserum for coating, not whole, unprocessed Na2HPO4, 2H2O 1.34 g
antiserum. NaCl 8.47 g
H2O ad 1 litre - check pH (7.2 ± 0.2)
b. The protein concentration of diluted antibody should be about 10 mg per L (optimal
concentration). Store at 4 °C. Stable for 2 months. (12 mL of buffer A is needed for one microwell plate).
c. Coating at room temperature for 1 hour is possible, but coating at 4 °C overnight increases
sensitivity and precision of the assay.
B. Washing Buffer, 0.01 M Phosphate Buffer, 0.50 M NaCl, 0.1% Tween 20, pH 7.2
d. Dilution of the antibody in buffer A gives ELISA results comparable to those obtained with the Dilution Buffer
commonly used 0.05 M carbonate buffer, pH 9.6. NaH2PO4, H2O 0.35 g
Na2HPO4, 2H2O 1.34 g
e. Microwell plates may be precoated and stored at 4 °C for several months, if the coating is NaCl 29.22 g
performed as follows: Dilute the antibody in buffer A and make the solution 0.01% in Tween 20
thimerosal. Add 100 µL antibody to each well and store the plate wrapped in plastic film at (Merck, No. 822184) 1 mL
4 °C. H2O 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).
Step 2 Washing
C. Chromogenic Buffer-containing
The 0.1% Tween 20 in the washing buffer reduces the background and should be preferred
Substrate OPD tablets of 2 mg 4 (DAKO OPD Tablets, Code No. S 2045)
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 H2O 12 mL
must be collected for disinfection. Allow the tablets to dissolve, then add
30% H2O2 5 µL

Step 3 Incubation with Test Samples Prepare the reagent shortly before use. Remember that the chromogenic substrate should
have reached room temperature before use. After the addition of H2O2 the reagent should be
a. Generally, biological samples should be diluted at least 1+1 to reduce the risk of non-specific watched a few minutes to ascertain that it remains slightly yellow. If the reagent colours
reactions. increasingly, it has been contaminated with peroxidase from unclean glassware or other
source.
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 The solution is stable at 24 °C for at least 2 hours if protected from light. The given volume is
improved if the sample is made 3% in polyethyleneglycol MW 6000. Further, the sensitivity suitable for one microwell plate.
will be improved by prolonging the incubation time, for example by incubating the plate - Note: If only OPD tablets without buffer salts are available, the H2O should be replaced by a
covered with plastic film - at 4 °C overnight. 0.1 M citric acid-phosphate buffer, pH 5.2, prepared as follows: citric acid,H2O 7.30 g,
Na2HPO4,2H2O 13.71 g, H2O ad 1 litre. Store at 4 °C. Stable for 2 months.
Step 5 Incubation with Peroxidase-Conjugated Antibody

a. It might speed up the reaction and improve the sensitivity if the peroxidase-conjugated D. 0.5 M Sulphuric Acid 28 mL 95 - 97% H2SO4 is mixed into about
antibody is made 3% in polyethyleneglycol MW 6000. In addition, the sensitivity might be 900 mL distilled water.
improved if the incubation time is prolonged 1 or 2 hours.
Note: Do not mix the water into the concentrated acid (risk of boiling).
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. Adjust the volume to 1 litre with distilled water. (About 15 mL of reagent D is needed for one
microwell plate).