Bead Plate: easy bead-based assays on a 96-well filter plate
Easy Bead-Based Assay Steps
In a typical bead-based sandwich assay using a Bead Plate (also known as Zoom Plate), functionalized beads dressed with capturing agent, i.e., capture antibody, are fist mixed with the sample and the detection agent, i.e., detection antibody conjugated with AP, HRP or fluorophores, in a separate container. After a desired incubation time, 10-20 µl of the bead mixture is added into a pre-blocked Zoom Plate and rinsed with 50 µl rinsing buffer four times. For fluorescent detection, the plate is ready for measure. For chromogenic and chemiluminescent detection, 10 µl enzyme substrate is added into each well and the signal can be measured within 15 min.
|Application Example: Bead-Based IL6 Fluorescent Sandwich Assay|
Fast and Sensitive Bead-Based Sandwich Assay Using Bead Plate. A 10-fold serial dilution of the human IL6 was prepared using the PBST-1%BSA dilution buffer. For each sample, 10 µl IL6, 10 µl 20 µm diameter agrose beads conjugated with anti-IL6 capture antibody, and 1 µl 100 nM AF647-anti IL6 detection antibody were mixed together in a 1.5 ml centrifuge tube. Multiple reactions were started simultaneously. After 30 min incubation at room temperature, 20 µl of each reaction mix was added into a Zoom Plate. After the solution was absorbed, agarose beads settled in the detection zone. To rinse, 50 µl rinsing buffer was added into the well and this was repeated for 4 times. The plate was then inserted into a fluorescent microplate reader for signal detection. Error bars are standard deviations of three samples.
Q: What's the selection guide for Bead Plate?
: Bead Plate can be used for a wide array of assays using different kinds of microspheres, such as silica beads, latex beads, magnetic beads, agarose beads and polyacrylamde beads, as long as the size of the beads are larger than the pore size of the filter membrane. We recommend using Zoom Plate with 2.7 µm pore size glassfiber membrane (cat# BGF27B and BGF27W) for beads larger than 4 µm, and Bead Plate with 1.6 µm pore size glassfiber membrane (cat# BGF16B and BGF16W) for beads larger than 2 µm. Membrane with larger pore size can provide faster flow rate and better rinsing. White plates are good for color display and can enhance luminescent signals, while black plates are ideal for chemiluminescent and fluorescent detection with sensitive detection instruments.
Q: Can 200 µl rinsing provide a clean background?
: Yes, for a total bead volume less than 2 µl and a regular dilution of labeled antibody (1:1000 or more, < 1 µg/ml), using 5 x 40 µl or 4 x 50 µl rinsing in a Zoom Plate well can provide a background as clean as conventional rinsing by multiple times of centrifugation and aspiration.
Several factors contribute to the low non-specific binding and high rinsing efficiency:
(1) The materials used for Bead Plate well and the absorbing plug are carefully-selected and tested low fouling polymer materials;
(2) If instructions are followed, the filter membrane and the portion of the absorbing plug in contact with the filter membrane is fully blocked before adding the beads into the Zoom Plate;
(3) Rinsing buffer is added immediately after the bead mixture and is absorbed away instantly, therefore the duration of the labeled antibody at the filter membrane is very short;
(4) Assuming the bead volume is 1 µl and holds equal volume of solution around it, adding 1 µl of rinsing buffer can reduce the concentration of unbound antibody to 50%. By removing this 1 µl of rinsing buffer and adding another 1 µl, the concentration of the unbound antibody will be reduced to 25%. Following this pattern, continuously adding 10 µl of rinsing buffer will reduce the antibody concentration to (1/2)^10, with is around 1/1000. So theoretically, rinsing with only 30 µl of rinsing buffer in a Zoom Plate can result in 10^9 reduction. (On the other hand, rinsing 1 µl of bead by centrifugation with 10 µl of rinsing buffer reduce the antibody concentration to 1/10 , while 200 µl of rinsing buffer can only lower the antibody concentration to 1/200.) This is the power of flow! Of course we should consider other factors that will come into play and reduce this theoretical efficiency, such as diffusion and the low but existing non-specific adsorption on the well. If you have a automatic or programmable pipette, multiple injections of small amount of rinsing buffer will generate even better result than 5 x 40 µl rinsing, which is suitable for manual pipetting.
Below is an experiment comparing rinsing in Bead Plate and rinsing by centrifugation. The result shows that after 4x40 µl rinsing, beads in Bead Plate has the same level of background signal as the beads rinsed with 4 times centrifugation and aspiration using 200 µl buffer each time.
|Thorough and Efficient Bead Rinsing. 1 µl agarose beads (20 µm diameter) conjugated with BSA was mixed with 5nM AF647-labeled mouse IgG in a total volume of 10 µl. The mixture was rinsed with PBST in black Bead Plate wells with glassfiber membrane 2.7 µm pore. Fluorescent signals measured after each 40 µl rinse (blue) is compared with that of the mixture rinsed with 4 times of centrifugation and aspiration using 200 µl PBST each time (red) , and the background signal of the same amount of agarose beads (grey). Error bars are standard deviation of 4 independent samples. The effect of 4 or 5 times of 40 µl rinsing in Bead Plate is equivalent to 4 times centrifugation rinsing.|
Q: After rinsing, the unbound molecules are contained inside the Plate. Will they interfere with the background signal?
: Not if the signal is detected within 20 min after rinsing.
Due to capillary force, the unbound molecules are wicked away from the detection zone and retained near the bottom the absorbing plug.
For fluorescent detection, the dense fiber material of the filter membrane and the absorbing plug blocks most of the excitation light shining from the top.
For chemiluminescent detection, the distance between the unbound molecules and the detection zone keeps them from reacting with the small amount of ECL substrate added onto the beads.
Of course, the unbound molecules will eventually diffuse back to the top is left for too long. According to our tests (see data below), fluorescent detection made within 0-20 min after the rinsing will not be affected by the diffusion effect.
Change of the fluorescent signal of the beads after rinsing. 1 µl agarose beads (20 µm diameter) conjugated with BSA was mixed with 5nM AF647-labeled mouse IgG in a total volume of 10 µl. The mixture was rinsed with 5x40 ul PBST in a Bead Plate (cat# BGF27B). After rinsing, fluorescent signals of the bead and Ab-AF647 mixture (blue) and the same amount of control BSA-agarose beads (red) were measured every 10 min. Error bars are standard deviation of 4 independent samples. The fluorescent signal of the beads inside the Bead Plate is stable within 30 min after rinsing. Afterwards, fluorescent intensity slowly increases due to diffusion of the unbound AF647-labeled antibody.
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