Elisa

ELISA is frequently run on an automated system because it has many time-consuming steps including the addition of reagents, performing timed incubations, and subsequent plate washings before reading the plate in a plate reader. Most ELISA assays produce a color or fluorescence end product that, when the reaction is stopped, gives an end point reading. A robotic system is an ideal platform to perform ELISA assays because the forward scheduling software assures that the robot will perform the time-critical steps within the allowable time tolerance, so all steps are performed in exactly the same manner. The steps in a typical ELISA assay include (1) transfer of samples to be tested to an antigen-coated plate, (2) addition of reagent #1 (conjugate), (3) shaking, (4) incubation, (5) washing, (6) addition of reagent #2 (substrate), (7) shaking, (8) incubation, (9) washing, (10) addition of reagent #3 (stop solution), (11) shaking, (12) reading the plate in a plate reader.

A robotic system for an ELISA assay will need the basic system consisting of robot, servo track, gripper for micro plates, plate turning station, reagent dispenser (three different reagents are to be dispensed with minimum washing between reagents), liquid handler, incubator, plate washer, plate shaker, plate reader, and plate bar code reader. Separate locations in the plate storage unit will be dedicated to the sample plates and to the coated assay plates. The operator will load the sample and coated plates, refill the reagent reservoirs, replenish other consumables, and input the various run variables. Variables can include the number of sample plates to process, the volumes of reagents to be dispensed, the incubation temperature and times, the wavelength at which the plate should be read, etc. The robot will select the first sample plate from the plate storage unit, read the bar code if applicable, and place it onto the liquid handler. The samples will be transferred from the sample plate to the coated plate, with the pipetting tip being washed between samples and the original sample plate returned to the plate storage unit or placed in the waste container. The coated sample plate will be processed with the successive addition of reagents, incubations, and washing until it is ready to be read.

When the plate is read in the plate reader, the output from the reader will be a file of values, one for each plate well (96 values for a 96-well plate or 384 values if a 384-well plate is used). The file will be captured and stored by the robotic system controller. Although the samples to be tested are transferred from the bar coded sample plate to the antigen-coated assay plate, which may or may not be bar coded, third-generation software can track samples from plate to plate without bar code identification on each plate. The system controller merely remembers the position of the subsequent plates and transfers a virtual identifier to the subsequent plates, always knowing the location of any group of samples.

The readings from the plate reader will be linked with the identification (ID) of the original sample plate and can be outputted to a company-wide Laboratory Information Management System (LIMS). The data can also be reduced with reagent factors from the reagent manufacturer and presented in a spreadsheet format. Assay results are displayed with the plate bar code ID, and the resulting ''plate map'' shows the well locations for the samples, standards, controls, and blanks. With the robotic system's multitasking software and forward scheduling capabilities, multiple plates can be in process at one time. This ensures faster sample throughput, less robot waiting time, and higher system utilization.

2. Cell-Based Assays

Cell-based assays may use the same basic robotic system as described above, with the addition of a plate lid park station and a 37°C CO2 incubator. Tissue culture plates are compatible, as the plate covers are easily removed to allow access to the plate wells, primarily for pipetting, and are easily replaced. Although the robot could simply remove the plate lids and place them on a surface while liquids are transferred to the well, the lid could be easily contaminated from the resting surface. A better approach is to suspend the plate cover so it does not touch any surface. One novel approach is to hold the cover from the top by vacuum, then releasing the cover back onto the plate. The first cover holding position can be above a hole in the table top with a waste container below it, so covers can drop directly into the waste when the cover is not needed for the balance of the assay. A typical robotic lid holding device is shown in Figure 7.

A practical design 37°C CO2 incubator provides one access door for the robot on one side and another access door on the other side for the operator. If there is more than one set of incubation parameters (temperature, humidity, or CO2) in an assay, then multiple incubators would be used. While it is possible for the system controller to change the temperature or CO2 concentration remotely, it can take one or more hours for the incubator to reach the new conditions, which is not quick enough for a running assay.

3. Enzymatic Assays

Enzymatic assays are difficult to generalize and there will be customized differences for a specific enzymatic assay [8]. One enzymatic assay, for cytosolic phos-pholipase A2 (cPLA2), is simple to perform on the basic robotic system described above. The steps include addition of 14C labeled substrate in a microplate, addition of the compounds to be tested, addition of cPLA2, and incubation for 30 min at 37°C. Reaction is stopped by the addition of a quench reagent, extracting the cleaved fatty acid, and separating the top organic layer in each well with an HPLC. The 14C labeled fatty acid is detected by a flow scintillation counter.

Tyrosine Kinase Autophosphorylation Assay. This assay is useful in investigating the signal transduction process and is measured using a scintillation counter [9]. Coated plates are coated with antiphosphotyrosine monoclonal antibody, incubated at room temperature in the plate storage unit, and then washed with a standard plate washer. Appropriate reagents and sample can be added to the plate with either the liquid handler or the 96-channel pipettor. The plate is then incubated at room temperature, the liquid aspirated and discarded, and the plate washed again. The plates are then air dried, the scintillating reagent is added to each well with either the liquid handler or the 96-channel pipettor, and the plate is read in a scintillation counter. This assay can be performed with the basic robotic drug screening system using a scintillation counter as the plate reading instrument.

4. Receptor Binding Assays

Filter Binding Assay. Filter binding assays for receptor/ligand complexes are widely used in the drug discovery process; they utilize a plate harvester to transfer the cells from the assay plate to the filter plate. The filter plate is then

Figure 7 A typical plate lid storage device holding microplate lids from overhead by vacuum.

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