Sometimes with ELISA performed in a MicroWell plate unexpectedly higher(or lower) optical
densities (O.D.) are measured in the peripheral wells than in the central wells. This
phenomenon is called ”edge effect”.
The most probable causes of this effect are illumination or
temperature differences between the peripheral and the central wells.
Light may cause edge effect if the substrate is photosentitive
(i.e. converted by light exposure) like the H2O2/OPD substrate in the peroxidase system. Thus, if strong light is
coming from one side (e.g. sunlight from a window) during the substrate reaction, the
peripheral wells closest to the light source may give elevated O.D. values.
Temperature difference, however, is the most common cause of edge
effect.
Incubation at 37°C instead of room temperature is often used for shortening incubation
times due to the fact that at higher temperatures the dissolved molecules move faster and
will therefore reach the well surface sooner than at lower temperatures.
However, a common mistake is to use reactant liquids straight from a
refrigerator and then incubate in a 37°C incubator (or at room temperature). Temperature
changes of these magnitudes may, especially with short incubation times, destroy the assay
homogeneity in MicroWell plates. The peripheral wells will normally be heated up first
because of their position closest to the lower edge of the plate, which is in direct
contact with the warm incubator shelf. Therefore, more reactant molecules may be
immobilized in the peripheral wells, which may result in higher O.D. values in these
wells, other things being equal. The edge effect may be more pronounced if plates are
stacked during incubation, especially in plates in the middle of the stack because their
central wells are shielded from the warmer surroundings by the plates above and beneath.
To demonstrate a pronounced edge effect caused by temperature differences, a stack
of 5 plates with 4°C IgG:peroxidase conjugate, 200 µl per well, were incubated at 37°C
for 30 minutes prior to substrate reaction. All the plates showed edge effect compared
with a control plate with room temperature conjugate incubated at room temperature. The
most pronounced effect was observed in the second bottom plate, the results of which are
given in Table 1 and Fig. 1.
Even if temperature changes are avoided, a small temperature dependent
edge effect may remain, which can be disturbing in critical assays when incubation times
are short. Due to heat consumption by evaporation (which is assumed to be equal from all
the wells in uncovered plates), the wells will be cooled down. However, the heat loss will
be restored faster in peripheral wells than in central wells, thus producing temperature
differences and possibly edge effect.
To avoid the above-mentioned problems, the following precautions should
be taken:
- Incubations should take place in subdued light or in the
dark.
- Reactant liquids (and plates) should be adjusted to the
temperature intended for incubation.
- Plates should be sealed with adhesive tape or placed in a
100% relative humidity environment during incubation.
Peter Esser
Nunc Laboratories
Further reading:
Burt, S. M., T. J. N. Carter and L. J. Kricka (1979), J. Immunol.Meth. 31, p.p. 231-236.
Oliver, D. G., A. H. Sanders, D. Hogg and J. Words (1981), J. Immunol. Meth. 42, p.p.
195-201.
Løvborg, U., Guide to Solid Phase Immuno Assays (1984), Nunc, Denmark. |
