Membranes are widely used to bind and detect nucleic acids and proteins. The use of these membranes in a 96-well filter plate format allows researchers to bind biomolecules and detect specific interactions with marker molecules simultaneously in a large number of samples. BioTrace NT (nitrocellulose) and BioTrace PVDF (polyvinylidene fluoride) membranes strongly bind biomolecules such as DNA and protein. Once specific molecules are bound to the membrane in the bottom of the well, further non-specific binding can be prevented through the use of a variety of blocking agents.

Following the blocking step, the filter plate can be incubated together with a labeled probe or antibody, allowing specific binding and hybridization interactions to take place in the plate. After the filtration washes remove the unbound probe/antibody, the AcroWell 96 filter plates can be read directly using a variety of marker systems in most detection devices.

Applications using the AcroWell 96 filter plate to bind and detect biomolecules include:

• ELISA
• ELISPOT
• Kinase assays
• Nucleic acid hybridization
• Molecular diagnostics
• Cell-based immunodiagnostics
• Protein:protein and protein:nucleic acid interactions

The AcroWell 96 filter plate consists of two parts, a clear polystyrene lid and a chemically resistant/biologically inert polypropylene filter plate assembly. The AcroWell 96 plate’s rigid single-piece construction meets the Society of Biomolecular Screening’s (SBS, Danbury, CT, USA) design recommendations and can be used in most robotic systems. The plate has two membranes sealed to the bottom of each well using a patented process that minimizes crosstalk and weeping (see Scientific and Technical Report, The AcroWell Filter Plate Minimizes Crosstalk). The upstream membranes, either BioTrace NT or BioTrace PVDF, are biomolecule-binding membranes with nominal pore sizes of 0.2 and 0.45 µm, respectively. The downstream membrane support layer is Emflon® membrane (hydrophobic PTFE) which protects the binding membrane layer and acts as a barrier to passive flow, allowing longer incubations with the wells filled with hybridization and immunodetection solution. The small hole in the bottom membrane support layer of each well allows fluid to pass under applied vacuum or centrifugation.

Vacuum Filtration: The recommendations of the SBS were key considerations during the design of the AcroWell 96 plates. As a result, the plate does not require “custom” hardware and can be used with most commonly available stand-alone and robotic manifolds. Place the device on the vacuum manifold and apply vacuum. [Recommended vacuum is 25.4 cm Hg (10 in. Hg); do not exceed 38.1 cm Hg (15 in. Hg)]. Most house vacuum and aspirators do not exceed 38.1 cm Hg (15 in. Hg). Release vacuum from the manifold. Do not release vacuum by pulling the corner of the plate as this can degrade the manifold gasket. Gently tap the plate to remove any hanging droplets.

Centrifugal Filtration: Place the AcroWell 96 plate on top of a receiver plate and insert into a standard rotor assembly for microtiter plates. Ensure that the rotor is properly balanced to prevent excessive vibrations. Centrifuge at 500 x g for one to two minutes.

PVDF Wetting Protocol: BioTrace^™ PVDF membrane is intrinsically hydrophobic and will not wet with water directly. It can easily be pre-wet by filtering a polar solvent such as methanol, isopropanol, or 70% ethanol. After filtering 100 µL of a polar solvent, an aqueous buffer can be added to the well and filtered to remove the residual polar solvent. Plates that are to be used within 30 minutes will remain moist. If the pre-wet plates are not used within 30 minutes, add water or buffer to each well. Plates that are allowed to dry will require the addition of polar solvents for re-wetting.

High Temperature/Long Incubations: Hybridization and antibody binding steps often require extended or high temperature incubations. The AcroWell 96 filter plate was designed to prevent weeping of solutions for short incubations, but some weeping may occur during incubations that are lengthy and/or at elevated temperatures.

Our studies have shown that very little or no weeping occurs at either three hours at room temperature or for overnight at 4 °C incubations. When performing overnight antibody binding incubations, it is recommended that a receiver plate be placed under the AcroWell 96 filter plate to prevent drops from falling and contaminating plates stacked below. Gently tap the filter plate over the receiver plate to remove any hanging drops.

The AcroWell 96 plates with BioTrace NT and PVDF membranes can be used at hybridization temperatures up to 65 °C without adverse effects. Overnight, high-temperature incubations containing detergents may weep approximately 10 µL per well without adversely affecting results. During hybridization, the plate should be placed on top of a solid-bottom receiver plate and sealed in a bag containing a moist paper towel. At the end of the incubation, gently tap the filter plate over the receiver plate to remove any hanging drops and avoid the chance of crosstalk.

Number of Washes: Repeated wash steps do not appear to remove previously bound biomolecules, as no significant difference in signal was observed when plates with bound antibody were washed two, three, and four times (Figure 1).

Figure 1 Effects of Multiple Washes on Europium-labeled Antibodies Europium-labeled antibodies (DELFIA*) were bound to AcroWell 96 filter plates with BioTrace NT and BioTrace PVDF membranes following the addition of increasing amounts of protein-blot blocking solution. After labeled antibody treatment, a 200 µL PBS wash step was filtered at 25.4 cm Hg (10 in. Hg) for a total of two, three, and four washes. After washing, 100 µL of Europium Enhancement Solution (Wallac Oy, Turku, Finland) was added to each well, incubated five minutes, and signal was quantitated using a VICTOR* Multilabel Counter (PerkinElmer, Wallac) using time resolved fluorescence settings. Error bars indicate standard deviation, n = 8. TRF = Time Resolved Fluorescence, CPS = Counts Per Second, BKG = plate background. The data indicate that high binding occurs when blocking agents are absent or when inadequate concentrations of blocking agent are used. In contrast, increasing blocking agent completely blocks antibody binding, resulting in signals near plate background. Increasing the number of washes from two to four does not significantly alter signal strength at high or low antibody levels.

Optimizing Blocking Solutions: It is important to optimize any blocking solution in order to ensure that enough blocking agent is added to prevent non-specific binding. However, at high concentrations some blocking agents may foul the filter, preventing the flow of solution through the membrane and interfering with subsequent washes. A simple optimization procedure is described below and is based on the data reported in Figure 2.

Figure 2 Optimization of Blocking Solutions Based on the observations made in Figure 1, three washes were performed for all blocking optimization studies. Four different protein-blot blocking solutions were added to wells, incubated five minutes, filtered, and rinsed with 200 µL buffered saline (PBS). The same concentration of fluorescein- and Cy5-labeled antibodies were added to each well (except for negative controls), incubated five minutes, and washed three times with PBS. The signal was quantitated using a VICTOR* Multilabel Counter (PerkinElmer, Wallac) at Cy5 and Fluorescein wavelengths. Error bars indicate standard deviation, n = 4. CPS = Counts Per Second; Blotto A (DIG High Prime DNA Labeling and Detection Kit Blocking Solution, Boehringer Mannheim, Germany), Blotto B (Milk Diluent/Blocking Solution Concentrate, Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) = milk-based blocking agents; BSA = 0.5% Bovine Serum Albumin in PBS. The data show that all blocking solutions are not equally effective at preventing the binding of labeled antibody which is added following a blocking treatment. The polypropylene membrane in the AcroWell 96 plates with GHP membrane (included as a control) is extremely low biomolecule binding. As expected, the signal on the GHP control plates remained near background regardless of blocking agent presence.

 

1. Set up a two-fold serial dilution series in phosphate buffered saline (PBS) of the respective blocking solution starting at 1/128X and ending at 1X. Use one well(s) as a negative control (no block) and one well(s) as a positive control (no antibody).
   
   
2. Add 200 µL of the blocking dilutions to one or more rows on the AcroWell 96 filter plate. Incubate at room temperature for five to 15 minutes.
   
   
3. Filter at 25.4 cm Hg (10 in. Hg). The filtration may take several minutes due to the viscosity of the blocking agents. Once filtration is complete, add 200 µL PBS and store blocked plate with PBS until ready to use (one to two hours). Filter to remove PBS prior to antibody challenge.
   
   
4. Dilute labeled antibody in PBS to a concentration where the signal can easily be detected (usually between 20 to 100 ng depending on labeling efficiency).
   
   
5. Add 100 to 200 µL of the dilute antibody solution directly to the AcroWell 96 plate with BioTrace NT membrane or pre-wet AcroWell 96 plate with BioTrace PVDF membrane (see PVDF wetting protocol above).
   
   
6. Incubate at room temperature for 5to 15 minutes and filter the dilute antibody solution through the AcroWell 96 filter plate (see above).
   
   
7. Add 200 µL of PBS to each well, filter, and repeat wash two to four times.
   
   
8. Add detection solution if needed and place the plate into the detector.
   

Each well in the AcroWell 96 filter plate can effectively bind up to 250 ng protein or antibody prior to blocking (Figure 3). This binding activity was consistently shown regardless of the labeling and detection system used. Variations in binding may be seen because of the high degree of variability in size, charge, and hydrophobicity. Most working concentrations of target proteins are well below these saturation concentrations.

The primary reason for determining the binding and blocking characteristics is to provide general guidelines for setting up experiments to detect specific binding. The data in Figure 4 show dilutions of a primary antibody bound to an AcroWell 96 plate with BioTrace NT membrane and blocked with an optimized concentration of a protein-blot blocking agent. This bound and blocked plate was incubated with antibodies having specificity to the primary antibody at constant concentrations in each well. After washing away unbound secondary antibody, the signal measured by the detector correlates well to the changes in concentration of the primary antibody.

Figure 3 Binding of Antibodies to Various Membranes Dilutions of Cy5-, Europium-, or fluorescein-labeled antibodies were added to wells that were either pre-wet (PVDF) or dry (GHP and NT). Antibody dilutions ranged from 1000 ng to less than 1 ng; the individual ranges were selected based on manufacturers’ recommendations. Aliquots of 200 µL of each dilution were added, incubated for five minutes at room temperature, and filtered at 25.4 cm Hg (10 in. Hg) vacuum. A total of three 200 µL PBS washes were followed by the addition of Europium Enhancement Solution to Eu-labeled plates or read directly in a VICTOR* Multilabel Counter (PerkinElmer, Wallac). Error bars indicate standard deviation, n = 8. Inset graphs show the logarithm plots for the BioTrace™ NT and BioTrace PVDF membrane-containing plates. TRF = Time Resolved Fluorescence, CPS = Counts Per Second. The data show that the AcroWell 96 plates with BioTrace NT and BioTrace PVDF membranes reliably bind 1000 ng protein per well. Saturation binding kinetics was seen at concentrations greater than 1000 ng labeled antibody (not shown). Antibody binding was resistant to washing and showed a linear correlation over two to three orders of magnitude (see inset logarithmic plot). The AcroWell 96 plate with GHP membrane, which is very low in binding, bound very little labeled antibody even at high concentrations.

Figure 4 Detection of Specific Binding Activities Unlabeled goat anti-dog IgG was added to AcroWell 96 plate with BioTrace NT membrane wells using dilutions from 400 ng to 250 pg total protein per well then incubated for five minutes and washed with 200 µL PBS. Optimized blocking was achieved with a 1/5 dilution of a milk-based blocking solution (blotto A), incubated five minutes, and washed with 200 µL PBS. This bound and blocked plate was incubated overnight at 4 °C with antibodies (Rabbit anti-goat) having specificity to the primary antibody (50 ng per well). The bound plates were then washed two, three, and four times with 200 µL PBS and counted using a VICTOR Multilabel Counter (PerkinElmer, Wallac). Error bars indicate standard deviation, n = 16. CPS = Counts Per Second. The specific binding-activity measurements are a combination of labeled antibody’s binding affinity for the target and the elimination of labeled antibody binding directly to the plate or membrane. The signal measured from the secondary antibody above correlates directly to the concentration of the primary antibody indicating effective binding of the target and adequate blocking. No significant difference in specific activity was detected for two to four washes. The AcroWell 96 plate with GHP membrane was used as a control because it is low in biomolecule binding and did not show significant specific or non-specific binding.

How much vacuum can I apply during filtration? The AcroWell 96 filter plate will reliably filter samples at 25.4 cm Hg (10 in. Hg) vacuum. Vacuum should not exceed 38.1 cm Hg (15 in. Hg) because well integrity may fail.

Why should I use the AcroWell 96 filter plate for an ELISA test? The primary advantage of the AcroWell 96 filter plate is that wash steps are simplified and sample (target) loss is minimized. Classical ELISA protocols involve the attachment of biomolecules to a solid bottom plate, the reaction with a target molecule, and washing several times to remove unbound material. Washing on solid surfaces involves the use of a plate washer that flushes buffer in and out of the well. Problems with target loss due to turbulence have the potential to negatively affect data. Washing by filtration eliminates these problems and streamlines the wash process.

Why is hybridization in AcroWell 96 plates preferred to standard dot blotting? Standard dot blots allow the placement of a variety of sequences on a membrane followed by the addition of a single probe to the hybridization mixture. If multiple probes need to be tested, multiple blots are required which wastes valuable time, sample, and probe. Hybridization in discrete wells allows a small amount of probe to be used allowing for a number of different probes to be simultaneously processed. The AcroWell 96 filter plate’s low crosstalk (see Scientific and Technical Report, PN 33177) allows adjacent wells to contain different probe mixtures.

Is binding capacity a problem with the AcroWell 96 plate? No. The BioTrace NT and BioTrace PVDF membranes are able to bind large amounts of protein and DNA. Preliminary data for the binding of IgG indicate AcroWell 96 filter plates containing BioTrace NT and BioTrace PVDF membranes easily bind 1000 ng/well total protein.

Once the initial biomolecule is bound, can further binding be effectively blocked? Yes. Protein blotting solutions (blotto) can be diluted and effectively block the further non-specific binding of proteins. Each blocking solution is different and may have to be optimized for blocking. A good start to optimization is to make doubling dilutions of blotto starting at a 1X concentration. If a blocking agent fouls the filter, then use another blocking agent or dilution.

Do the plate materials of construction bind biomolecules? No. We make the AcroWell 96 filter plate from a biologically inert polypropylene.

Can I filter non-aqueous solution with the AcroWell 96 filter plates? No. The Emflon membrane layer will “wet-out” with organic solvents. Alcohols, such as methanol, can be used to pre-wet the membrane in the BioTrace PVDF membrane plate because exposure to the solvent is brief and it is washed out with aqueous buffer prior to using the plate. The return of the plate to an aqueous environment will allow the Emflon membrane layer to be used as a flow director once more.

How can I use the AcroWell 96 filter plate for extended and high temperature incubations? The AcroWell 96 filter plate is designed to reduce weeping, and the materials are resistant to high temperatures. The plate can be placed onto a solid-bottom receiver plate and sealed in a bag together with a moist paper towel. Weeping should be less than 10 µL per well.

Can I use the AcroWell 96 filter plate with non-radioactive detection systems? Yes. We have tested the AcroWell 96 plate with Cy5-, Fluorescein-, and Europium-labeled antibody systems, and all systems show linear quantitation over several orders of magnitude.

Can I peel the membrane off of the bottom for processing? No. The strong seal created with the polypropylene plate prevents the removal of the membrane from the assembly. Hybridization and detection can be performed in the plate itself or on membrane punches. The robotic compatibility allows most detectors to use the AcroWell 96 filter plate for direct quantitation of signal.

How can I reduce well-to-well variability? Because the removal of unbound label using filtration is generally more uniform than plate washing, well-to-well variation is less. If variability is still an issue, lower the number of cells used to achieve signal.

Can I use the AcroWell 96 filter plate with my robotic fluid handling systems? Yes. The AcroWell 96 plate was designed to meet the SBS footprint and well location specifications.

Does the AcroWell 96 filter plate work on all vacuum manifolds? Every SBS-compatible manifold we have tested works with the AcroWell 96 filter plate. To use the AcroWell 96 plate on manifolds containing a metal grid, simply remove the grid. It is not needed for the SBS-compatible AcroWell 96 plates and can create problems with vacuum sealing.

Can I use the AcroWell 96 filter plate with robotics and robotic filtration stations? Yes. It was designed in accordance with SBS standards, making it reliable for most dispensing stations and compatible with most available robotic filtration stations.

Can the AcroWell 96 filter plate filter solutions once the membrane has dried out? BioTrace NT membrane is intrinsically hydrophilic and does not contain or shed surfactants, so it will easily wet-out and filter each time. BioTrace PVDF membrane will require re-wetting if dried (see general handling procedures). After wetting with polar solvent, it is advised to rinse with water; if the plate is not to be used right away, leave water in the wells.

The AcroWell 96 filter plate has a single membrane coupon. Is there a problem with well-to-well crosstalk? Pall’s patented sealing process eliminates the lateral flow of liquids between wells (see Scientific and Technical Report, The AcroWell Filter Plate Minimizes Crosstalk).

Can I use the AcroWell 96 filter plate to capture filtrate? Yes, if you use a centrifuge for filtration. Collection of filtrate from vacuum-filtered samples is not recommended.

Can I filter organic solvents with the AcroWell 96 filter plate? We do not recommend this because the Emflon membrane support layer will wet-out and crosstalk may occur.

Can I use the AcroWell 96 plates with radioactive detection systems? Yes. After processing and binding the labeled sample, scintillation cocktail can be added to the plate. Because AcroWell 96 plates are low weeping and robotic-compatible, they can be placed directly into a variety of detectors.