Reprinted with permission of Nalge Nunc International
In Nunc Bulletin No. 1 (second edition 1997) circumstances which can cause positive edge effect were discussed.
Here we will discuss negative edge effect, i.e. unexpectedly lower optical densities in peripheral wells than in central wells of a MicroWell matrix, due to temperature differences.
If, during some incubation in ELISA, one of the six conditions listed in Table 1 occurs, an edge effect of the corresponding sign (positive or negative) may be observed. It appears from Table 1 that the sign of the edge effect is dependent on the relative temperature of the surroundings, negative edge effect being the case when the surroundings are colder than the reactant liquids and/or the wells per se. An exception would be in "competitive" ELISA, where sample and labeled standard are incubated successively, starting with the sample. In this case relatively cold surroundings would give positive edge effect and vice versa.
Whereas positive edge effect easily occurs due to the short incubation time normally used in warm incubators (37°C), negative edge effect will be blurred due to the long incubation time normally used in cold incubations (4°C). Therefore, negative edge effect is a less common, or at lest less pronounced, phenomenon than positive edge effect.
However, if negative edge effect is observed or suspected in an assay, one should always consider the causative conditions listed in Table 1.
If one of these conditions occurs during a brief substrate reaction, the negative edge effect may be expressed rather easily. Especially, this is likely to occur if the incubation prior to substrate reaction is a relatively warm one, which may establish the last case mentioned in Table 1.
To demonstrate the negative edge effect in this case, a NUNC-IMMUNO® Plate MaxiSorp F96 was incubated with 200 µl/well of IgG:peroxidase conjugate at 37°C for two hours (plate and conjugate were both preheated to 37°C), immediately followed by quick washings (within one minute) and H2O2/OPD substrate reaction for 3.5 minutes at room temperature. The plate showed negative edge effect, as illustrated in Table 2 and Figure 1, compared with a plate which had been adjusted to room temperature before substrate reaction.
Obviously, to eliminate edge effects, not only the reactant liquid should be adjusted to the temperature intended for incubation, but also the wells per se.
Dependence of edge effect sign on temperature conditions during (at the beginning of) incubation.
RELATIVE TEMPERATURE OF | Sign of edge effect |
Reactant liquid | Wells | Surroundings (Incubator) |
Cold | Cold | Warm | Positive |
Cold | Warm | Warm | Positive |
Warm | Cold | Warm | Positive |
Warm | Warm | Cold | Negative |
Warm | Cold | Cold | Negative |
Cold | Warm | Cold | Negative |
O.D. readings of the H2O2/OPD reactions in a Nunc-Immuno MicroWell Plate after incubation with IgG:peroxidase conjugate at 37°C for two hours. (See text for further experimental details.)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | M | SD | CV |
A | 958 | 1043 | 1059 | 1045 | 1036 | 1040 | 1033 | 1035 | 1035 | 1030 | 1011 | 974 | 1025 | 29.8 | 2.9 |
B | 1020 | 1104 | 1108 | 1104 | 1093 | 1098 | 1067 | 1081 | 1068 | 1088 | 1044 | 1038 | 1076 | 29.0 | 2.7 |
C | 1025 | 1095 | 1099 | 1097 | 1096 | 1091 | 1044 | 1086 | 1080 | 1077 | 1066 | 1033 | 1074 | 26.3 | 2.4 |
D | 1037 | 1107 | 1118 | 1117 | 1141 | 1121 | 1100 | 1112 | 1118 | 1103 | 1087 | 1062 | 1102 | 28.3 | 2.6 |
E | 1029 | 1086 | 1088 | 1076 | 1087 | 1084 | 1084 | 1072 | 1066 | 1072 | 1054 | 1030 | 1069 | 21.0 | 2.0 |
F | 1026 | 1062 | 1095 | 1046 | 1080 | 1099 | 1058 | 1072 | 1063 | 1050 | 1070 | 1037 | 1063 | 21.9 | 2.1 |
G | 1021 | 1079 | 1107 | 1081 | 1091 | 1119 | 1072 | 1082 | 1087 | 1083 | 1068 | 1036 | 1077 | 26.9 | 2.5 |
H | 956 | 1036 | 1043 | 1059 | 1055 | 1042 | 1042 | 1034 | 1029 | 1019 | 1014 | 992 | 1027 | 28.8 | 2.8 |
M | 1009 | 1077 | 1090 | 1078 | 1085 | 1087 | 1063 | 1072 | 1068 | 1065 | 1052 | 1025 | 1064 | — | — |
SD | 32.5 | 27.0 | 25.8 | 26.8 | 30.9 | 30.9 | 22.8 | 26.2 | 28.4 | 29.4 | 27.2 | 28.2 | — | 35.6 | — |
CV | 3.2 | 2.5 | 2.4 | 2.5 | 2.8 | 2.8 | 2.1 | 2.4 | 2.7 | 2.8 | 2.6 | 2.8 | — | — | 3.3 |
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Block diagram of the plate O.D. readings listed in Table 2, illustrating the negative edge effect. Each column represents the O.D. reading of the respective well in percent of the plate mean value (1064 mEU). Note that the edge effect is most pronounced in the corner wells, A1 and H1 giving the minimum value = 90%, whereas the central well D5 gives the maximum value = 107%. |
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