Negative Edge Effect in MICROWELL ELISA

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.

Table 1

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

Table 2

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

Figure 1

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%.

Disclaimer: Cole-Parmer products are not approved or intended for, and should not be used for medical, clinical, surgical or other patient-oriented applications.