Resources - Technical Notes Using EL406™ and BioStack™ to Automate the Liquid Handling Steps of Assays - Semi-Automation of ELISA and Colorimetric AssaysDownload
January 15, 2009
Authors: Paul Held Ph. D, Senior Scientist, Applications Dept., BioTek Instruments, Inc.
Many biological assays such as ELISA or colorimetric assays have been standardized through the use of microplates, allowing the use of instrumentation to automate specific fluid movement tasks such as plate washing or the addition of reagents. Typically each task requires a dedicated instrument to accomplish the fluid handling task with manual intervention to move the plate to each instrument. The EL406™ is a multifunctional microplate washer and dispenser capable of carrying out the different fluid handling tasks for a single assay. Here we describe the details of using Liquid Handling Control™ (LHC™) PC software to control an EL406 in conjunction with a BioStack™ Microplate Stacker to automate the liquid handling portion of many assays.
Programming the Liquid Handling Control software (LHC) to automate multi-step processes is straight forward. LHC handles tasks in a linear fashion from the beginning of the protocol steps towards the end with no multitasking. Once a step has been completed, the next step is initiated. With this in mind, one can automate a series of liquid handling and incubation steps that are performed in succession for a series of plates.
Correct timing between steps is the paramount issue in regards to programming automated assays using LHC with the EL406 and BioStack. Differences in timing for process steps and process times with various plate counts need to be accommodated if repeatable and valid results are to be obtained. For example, the time it takes the EL406 to dispense 200 µL of a reagent would be greater than the amount of time required to only add 50 µL of another reagent. Likewise, the time it takes to process one step of an assay for 10 plates would be longer than the time required for 5 plates. These differences in process times between steps, which need to be accounted for in order to achieve optimal and repeatable results, can be compensated for by using the “Delay” command in LHC. This command is typically used under two circumstances. Primarily it is used to provide ambient incubations when all of the plates have been returned to the BioStack and then restacked. Note that the delay time used is dependent on the number of plates processed. Because the delay needs to start after all of the plates have been processed and subsequently restacked, one needs to calculate a different delay interval for different plate counts.
The equation for delay time is as follows:
Eq. 1 Dn= Inc – (Pn+Rn)
Where Dn is the delay time for n number of plates; Inc is the intended total incubation time; Pn is the process time required for n number of plates; and Rn is the time necessary to restack n number of plates. For example if the intended incubation time was 30 minutes, the preceding process takes 30 seconds per plate, the Delay time would be 26 minutes and 31 seconds for a 5 plate batch. Dn= 1800 seconds – ((5*30 seconds) + 59 seconds). Therefore Dn= 1591 seconds or 26 minutes and 31 seconds.
Figure 1. EL406 Microplate Combination Washer Dispenser and BioStack Microplate Stacker.
In addition to delaying the initiation of the next process step in order to provide the necessary incubation intervals, the delay command can be used to control the specific timing between EL406 procedural steps. With most ELISA process steps the actual procedure is a repeated task. For example, a dispense step on a microplate can be broken down into 12 separate dispense procedures; one for each 8-well strip of the plate. As dispense-volumes increase, the time to accomplish the procedure increases as well. This difference has to be accommodated in order for the correct assay timing to be maintained.
Figure 2. Avian Influenza Virus Antibody Assay Test Procedure Steps. Processes carried out by the EL406 Washer Dispenser are indicated in Red.
The differences between onset of incubation periods and the onset of delay steps is the most complicated feature of LHC automation programming. Incubation steps typically will begin at the completion of a process task on the first plate, while delay steps begin with the completion of the previous step on the last plate. In other words the incubation for plate-one would naturally begin after the addition of a reagent. For example, the Avian Flu assay requires 30 minute incubation after the addition of sample (Figure 2), which is a manual process. Note that the initial delay within LHC is for 29 min 04 sec rather than the 30 minutes suggested by the assay protocol (Figure 3 line 1). This difference is the result of the time required to prime the washer and three dispensers.
The second incubation, also 30 minutes, is accommodated with a 22 min 35 sec delay (Figure 3, line 13). The 7 min 25 sec difference is the time from the addition of conjugate to the first plate to the completion of the process of the wash-cycles, reagent addition and restacking of the five 96-well plates back into the input stack (see Table 1).
During that period of time, the first plate was essentially incubating at an ambient temperature, while the other plates were being processed. Once all of the plates are finished, they are restacked. With the completion of the delay, the fist plate is again transferred to the EL406 and the “Add Substrate” assay step is initiated.
The 2 second delay denoted in the “Add Substrate” step addresses the timing differential of adding reagent with the peristaltic pump rather than the syringe pump (Figure 3, line 18). Likewise the 32 second delay in the “Add Stop” step accommodates the difference in timing from the previous step due to the lack of a wash procedure (Figure 3 line 25).
Figure 3. LHC Process steps required to Automate Avian Influenza Antibody Assay for a five plate run.
The total time required for the EL406 to complete an assay process increases with the plate count, while the assay’s incubation time remains constant. For example, it takes more time to wash and add reagent to 10 plates than it does 5 plates, but the incubation time following that specific procedure in the assay would remain the same. The delay function of LHC is used to account for the difference between the process time and that of the incubation (see eq. 1). Because the process time changes with plate count, the delay period that is linked to the incubation steps needs to be changed accordingly for each differing plate count so that the total time used is equal to the incubation time.
This requires that different LHC files are created for each run that uses different numbers of microplates. In other words, a run that has 5 plates would require a LHC file and a run that had 10 plates would require a separate LHC program file. Each file would have the same process steps, but the delay time would be different to accommodate the different plate counts.
At some point the process time of the different assay steps needs to be determined experimentally. While it is easiest to time the process steps of a single plate and then multiply that value for the number of intended plates, the most accurate method would be to time a dummy run with the exact number of plates required. By using a stop watch to time each of the process steps one can calculate the delay time required
Table 1. Restack Time Requirements
1 min 55 sec
2 min 54 sec
3 min 53 sec
4 min 48 sec
5 min 45 sec
7 min 47 sec
9 min 34 sec
Loops: Should only be used for repetitive tasks by the EL406, such as a wash step and a dispense step that are repeated on the same plate before returning to the BioStack. While one could use a loop command followed by a BioStack command to define the exact number of plates to be processed, this is not recommended. The inadvertent use of more plates than outlined in the LHC program would result in plates not being processed. It is better practice to only put the number of plates to be processed in the input stack and use a “Use BioStack” command that in essence is a loop function to deliver and retrieve plates. This command will process all of the plates in the stack.
Long runs: Priming of peristaltic pump. The tubing of the peristaltic pump dispenser is permeable to gases. As a result over time air will diffuse through the tubing into the fluid lines resulting in bubbles forming in the fluid path. In order to eliminate this phenomenon, the dispenser needs to be primed periodically. It is suggested that a prime step occur every hour that fluid is in the tubing or that the prime step occur immediately prior to its use.
Comment lines: Use of the comment line command is a useful tool within the LHC programming to denote specific process steps. Not only do they assist during the programming of the assay, they can also be a useful tool to monitor the assay process during the run.