Flow-through immunoassays, also known as vertical flow or transverse flow immunoassays, are basically “semi-dry” dot-blots. Due to miniaturization and a certain degree of integration, the volumes of sample and reagents are reduced and the assay can be performed in a simple disposable device. The reaction membrane is laid on top of an absorbent pad. When agents are applied onto the membrane they are drawn through by the absorbent which acts as a sink for all liquids used in processing of the test.
Pros and cons of flow-through immunoassays
The degree of integration is considerably less compared to lateral flow immunoassays. This makes development and production easier and may also avoid some of the most important patents which apply to immunodiagnostic assays. It may also be easier to develop multi-analyte tests because the spatial position of a capture molecule on the membrane is not linked to the assay dynamics as in a lateral flow assay.
On the other hand, the lower degree of integration normally requires more complex protocols for processing the assay. Multiple additions of reagents may be required. Typically the sample is applied in a first step and the detection reagent, such as a colloidal gold conjugate, is added in a second step. Enzyme-linked detection systems may also require the addition of substrates. Sometimes an off-line sample preparation step is also necessary. Reading and interpreting the result may be less unequivocal and can be confusing for inexperienced users.
Nitrocellulose reaction membranes
In a flow-through assay the liquid passes vertically through the reaction membrane, so the filtration properties of the membrane are more important here than capillary rise times. In principle, all membrane types that have a high and consistent protein binding, could be used. However, for the same reasons than for lateral flow immunoassays — wettability, easy capture protein application, easy blocking, and reliability — nitrocellulose is again the material which is most frequently used. Small-pore unsupported membranes such as BA83 and BA85 can be used; they are highly sensitive small-pore membranes with large surface area and high protein binding capacity. However, they have to be carefully encapsulated, ensuring good contact between the membrane and the absorbent to give good flow.
- Pore size: smaller pore size equates to higher surface area for conjugates to rest on, and therefore higher protein binding capacity. However, this comes with a cost of slower flow rate
- Backing adds mechanical support to the membrane. Use of an unbacked membrane might be good enough depending on mechanical load of the sample.
The Whatman™ advantage
- The recommended Whatman™membranes are manufactured specifically for vertical flow assays to eliminate problems caused by capillary rise.
- Small pore structure provides low non-specific binding and greater sensitivity for accurate results.
- 100% pure nitrocellulose provides high binding capacity.
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The function of the absorbent pad in a flow-through assay is considerably different from that in a lateral flow assay. It must be in close contact with the entire lower surface of the reaction membrane to ensure a homogeneous flow. This is necessary to ensure consistent signal development. Typically, different agents with different properties are added sequentially. The total volume of the solutions added can be quite large. Thick cellulose materials with fast wicking and very high water absorption capacity are usually required.
The most important parameter of the absorbent pad in a flow-through assay is its water absorption capacity. The pad must be capable taking up all the liquid which is added during processing of the assay. Therefore, the test developer must choose a pad which can accept at least this amount of liquid. In extreme cases, this may require a stack of pads.
The absorbent media used for flow-through assays must wick rapidly and be highly water absorbent. The volumes of liquids used in flow-through assays can be much higher than those in lateral flow. Thicker cellulose materials with fast wicking are therefore the product of choice.
The Whatman™ advantage
- 100% pure cellulose for fast wicking and high water absorbency.
- Available in a range of thicknesses sufficient for most flow-through assays.
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