Prior to running a multiplex ELISA kit, ensure all equipment is functioning properly to obtain optimal results.
Pipettes should be regularly calibrated and maintained according to manufacturer’s specifications. Check pipettes between calibrations using gravimetric methods, such as weighing the maximum and minimum volumes of water four times each, using a new pre-rinsed tip for each volume setting, and ensuring that the mean volume and standard deviation for each volume setting meet manufacturer’s specifications.
Q-View Imagers can be calibrated by going to Settings > Administration > Manage Imagers. To calibrate alternate imagers, follow manufacturer’s instructions. Qualified customers can receive a FREE Calibration Kit specifically for imager validation purposes by contacting firstname.lastname@example.org.
Automatic Plate Washer
First, ensure that the plate washer has a program that will not scratch the bottom of the microplate and will preferably leave a small, uniform amount behind to prevent plate drying. For example:
|Process||Distance||“Steps” on a Biotek ELX-405|
|Aspiration Height||3.810 mm||30 (this leaves approximately 3 uL at the bottom of each well to help prevent plate drying)|
|Aspiration Position||1.28 mm from center||-28|
|Dispense Height||15.24 mm||120|
|no soak or shake cycles are needed|
Next, ensure that all washer pins are functioning: connect the prepared wash buffer to your automatic plate washer, run 1-2 priming cycles, and then separately dispense and aspirate 100µL wash buffer into a spare microtiter plate, and ensure that all pins dispensed or aspirated uniformly.
For all our multiplex ELISA kits, we recommend using a rotational microplate shaker capable of 300-1,100 rotations per minute (RPM), such as a Barnstead/Labline 4625 titer plate shaker, IKA MTS 2/4, or equivalent. Prior to running the assay, determine the shaker’s actual RPM using the manufacturer’s instructions. If no instructions are available, use one of the following protocols:
- To digitally determine the shaker’s RPM, secure an accelerometer (such as a smartphone with an accelerometer app) onto the platform, turn the shaker on for 10 seconds, and record the number of peaks read by the accelerometer and multiply by 6 to calculate the RPM at that setting. Repeat this procedure at least 3 times to ensure accuracy.
- To manually determine the shaker’s RPM, fasten a pen or marker to the side of your shaker, and set a timer for 10 seconds. Pull a sheet of paper horizontally under the pen for the 10 second interval, and drop the paper once the alarm sounds. Count the number of oscillations and multiply by 6 to calculate the RPM at that setting. Repeat this procedure at least 3 times to ensure accuracy.