Incubation plate rotation

The reaction between antigens and antibodies relies on their close proximity. During ELISA, this is affected by their respective concentrations, distribution, time and temperature of incubation, and pH (buffering conditions). In any interaction, the avidity of the antibodies for the particular antigen(s) is also important. Two types of incubation conditions are common (1) incubation of rotating plates (with shaking) and (2) incubation of stationary plates. These conditions affect the times and temperatures required for successful ELISAs and therefore discussed separately. 

We have already eonsidered stationary vs rotated plates. The eonelusion is that rotation of plates for ineubation steps is highly recommended to eliminate vicosity effects time differences and temperature effects, including edge-well differences caused when plates are stacked and incubated stationary. However, when a rotator is not available, provided that standardization of methods is used, stationary plate assays are not a problem. The following tips are helpful when incubating plates that are not rotated ... 

Add 25 pL of Cell Titer Glo to all white solid-bottom plates. Incubate the plate at room temperature for at least 1 h on a rotating platform. Read luminescence using a luminescence microplate reader (see Note 12). 

Make serial dilutions of 10°, 10-3, 10-6, and 10"9 of the phage in SM buffer, and spot 5-pL aliquots of each dilution onto the solidified top agar. Tilt and rotate the plate to allow each spot to spread to a diameter of approx 10 mm, and allow the phage suspension to dry into the top agar. Incubate the plate overnight at 37°C. The approximate titer of the library is then be estimated from the number of plaques at each dilution. 

Add 200 pL of each compound concentration into triplicate wells. Add 200 pL of 1% Triton-X in buffer as positive control into each of the triplicate wells as well as 200 pL of buffer as negative control, and incubate the plate for 4 h on a shaker (rotation frequency of 200 rpm) in an incubator (37°C, 5% C02, 90% humidity) (see Note 8). 

Desalting the MassEXTEND products with CLEAN resin is a crucial step with strong impact on the data quality. It is important that the resin particles stay in suspension during the 5-min incubation step and do not settle. A rotation where plates are turned upside down usually provides best performance. Increased incubation temperature is not recommended. 

The effect of rotating plates is to mix the reactants completely during the incubation step. Since the solid phase limits the surface area of the adsorbed... 

The effect of temperature also has implications when many plates are stacked during incubation since the plates heat up at different rates depending on their position in the stack. The wells on the inside may take longer to equilibrate than those on the outside, which has a direct effect on the diffusion conditions, which, in turn, affects die ELISA. This is negated by rotation because there is the same chance of molecular contact in all wells. 

Incubate the plate under rotation (best) at 37jaC for 1 h or stationary at 37jaC for 2 h. Wash the wells. 

Incubate for 1 h at room temperature (or under conditions you used to titrate the conjugate). Rotate the plate to... 

The incubation step to allow binding of mAb to antigen should be that used in the CBT. One hour at 37jaC while plates are being rotated is usually adequate. 

Roll tubes may be used as an alternative to plates. In these the sample is added to a tube containing molten agar which is then rotated rapidly on a mechanical roller until the agar sets. The tubes are incubated and counted. This method uses less agar than traditional pour plates and is widely used in the dairy industry. 

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