Technologies: Lateral-flow

Lateral-flow: test components

Lateral-flow components Sample pad Conjugate pad Detection conjugate 4. Nitrocellulose membrane Absorbent pad Test line Control line Plastic adhesive backing card

1. The Sample Pad

The sample pad is made of cellulose, glass fiber or other material where the fluid sample is applied to the lateral flow device and, if necessary modifies it to improve the results of the assay. This might be by modifying pH, filtering out solid components, separating whole blood constituents, adsorbing out unwanted antibodies or some other test specific variable.

For some applications, the sample pad should be pretreated by dipping it into a specific buffer containing a mix of a solution comprised of soluble proteins, surfactants/detergents, and other polymers. These allow for a steady flow and prevent nonspecific binding of sample components to the pad.

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2. The Conjugate Pad

The conjugate pad is made of a non-absorbent material such as fiberglass pad, polyester, rayon or a similar material. The conjugate pad is always of a synthetic material (at least when using a gold conjugate) to ensure the efficient release of its contents.

As its name implies, the assays detection conjugate is dried down and held in place here until a liquid test sample is applied to the sample pad. The liquid from the sample, by capillary action moves into the conjugate pad, re-hydrates the dry gold conjugate and allows the mixing of the sample with the conjugate. The complex of gold conjugate and analyte then moves into and up the membrane. Pretreatment of the conjugate pad helps to ensure the conjugate releases at the proper rate and enhances its stability. The pretreatment is performed in the same way as with the sample pad.

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3. Detection Conjugate

The signal reagent used in lateral flow tests have become much more varied as the technology advances. The vast majority of tests on the market can be read by the naked eye, but new tests are being developed which require an instrumented reader and offer the hope of improving test sensitivity or delivering quantitative or semi-quantitative results.  Test may use colloidal metals such as gold or silver, carbon, a visible or florescent dye, magnetic particles, enzymes, latex beads impregnated with visual or flourescent dyes, or a combination of these which are conjugated to either an antibody or antigen to generate signal. In early versions of lateral flow tests latex was the most prevalent conjugate used as a signal reagent, however colloidal gold is probably the most commonly used signal reagent in use today. Depending on what reagent is used as a signal reagent will affect whether a strip can be simply read visually or if it will require an instrumented reader.

The vast majority of the available assays contain gold, colored latex or another visually observable particle adsorbed with antibodies or antigens specific to the analyte being detected. When the strip is designed to be used with a reader, the conjugate may not be easily visible to the naked eye. If the strip will be read visually, the detection particle must be large enough to be seen but nut so large as to overwhelm the antibody (or antigen) conjugated to its surface through steric hindrance. These particles usually run from 10-100 nm in diameter, but there are always exceptions.

Conjugate is added to the pad in one of two methods, immersion or spraying.  In immersion the conjugate pad is submerged in the conjugate-protein suspension. In spraying the pad is coated using quantitative, directional aerosol dispenser which is somewhat similar to an inkjet printer. Spraying offers much more control of the conjugate application and prevents dilution and washing away of the pad pretreatment, but it also adds a significant capital expense and might increase the complexity of strip manufacturing.

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4. Nitro-Cellulose Membrane

 The nitrocellulose (NC) membrane consists of a very thin Mylar sheet coated with a layer of NC. The benefits of NC as an assay matrix are the reason why it so completely dominates the rapid test market. These benefits include low cost, capillary flow, high binding affinity for protein, ease of handling and cutting, as well as the ability of manufactures to varying thickness and components of the membrane to suit customer and market needs. NC binds proteins electrostatically through and interaction with the nitrate esters and the peptide bonds of the protein. The membranes binding capacity is ultimately determined by the available surface area. This surface area is determined by pore size, porosity (pore density), membrane thickness, and unique physical characteristics of that particular polymer. These factors also affect capillary flow rate which can also dramatically affect a Lateral flow test’s overall performance. If a strip flows too fast you may lose sensitivity, and if it flows to slow you lose specificity (and can increase background).

Despite the advantages, nitrocellulose has a number of limitations. These include imperfect reproducibility of performance within and between lots, shelf-life issues, flammability, extreme sensitivity to organic solvents, fragility (especially with unbacked membranes), and variable performance due to environmental conditions such as temperature and humidity.

As with many immunological based assays, blocking may be necessary to prevent nonspecific binding of sample and conjugate to the test lines and to limit background along the membrane. Blocking is also used to control flow rates and stabilize test and control-line proteins. The blocking process involves immersion of the striped membrane in an aqueous solution of proteins, surfactants, and/or polymers. The membrane is then removed, blotted, and dried.

Requiring blocking of nitrocellulose membranes is an unfortunate corollary to using this material. While the blocking process is necessary to prevent product variation during the product’s lifetime, it can add cost and complexity to manufacturing.

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5. Test and control reagent lines

The complex of gold conjugate and analyte then moves onto the membrane strip and migrates towards the capture binding protein, where it becomes immobilized and produces a distinct signal in the form of a sharp red line. A second line, a control, may also be formed on the membrane by excess gold conjugate, indicating the test is complete.

The standard for lateral flow tests is one test line and one control line are placed on the NC membrane. These are usually closer to the absorbent wicking pad than to the sample/conjugate pads. Some lateral flow assays may have more than one test line, but each additional test line greatly increase the complexity of development, and thus increases cost. The addition of test and control reagents is done simultaneously. This is often done with a dispenser that gently slides a soft capillary tube across the membrane.  dispenser pump releases a constant volume of the reagents down the length of the membrane.  This system is simple, easy to use, and low cost. They can be somewhat cumbersome in large scale manufacturing and many systems require a technician to constantly feed the nitrocellulose cards and to monitor reagent levels as well as the quality of the test and control lines.

An alternative method is a non-contact aerosol system. These sprayers dispense solutions in controlled ultrafine, ultra- small volume aerosols. These devices project very fine droplets of reagent onto the membrane and overlap the drops to create a continuous line.

Spraying offers much more control of the reagent application, but it also adds a huge capital expense and dramatically increases the complexity of strip manufacturing.

These devices are more appropriate in very large scale manufacturing or when a reader with tight tolerances will be used to analyze the lateral flow test strips.

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6. Absorbent Pad

The absorbent pad, also called a wick or wicking pad, pulls fluid off of the membrane to allow the capillary flow of the membrane to keep flowing in the proper direction and at the proper rate. If an absorbent pad isn’t used (or if it separates from the membrane so it is functionally absent), the sample and buffer will back flow down the membrane and could raise the background or possibly cause false positives. This can also occur if the absorbent pad selected for the volumes of buffer and sample involved is inadequate.
Most absorbent pads are made from non-woven, cellulose fiber sheets. These pads can be manufactured in a variety of thicknesses and densities to suit the needs of the assay.

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7. Plastic-adhesive backing card

Due to the delicate nature of the materials used in an ICS assay as well as the need to maintain a precise, direct contact between components to ensure proper reagent and sample flow a backing card of some sort is always necessary. Usually these are made pre-treated with pressure-sensitive adhesive selected for its stability in the assay and to insure it doesn’t leach chemicals that may interfere with results. A related concern for manufacturers is that the adhesive is strong enough to properly bind the materials to the card but that it also doesn’t flow too far in to them and inhibit the capillary action by reducing the available bed volume. The adhesive card is initially covered with a liner which may be pre-slit for easier assembly of test components.

Many materials are available depending on the needs of the assay platform and manufacture configurations of the diagnostic tests. The more common materials are: polystyrene, vinyl, polyester (clear or opaque), and Mylar.

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8. Laminate Cover Tape

The Laminate Cover Tape is an adhesive tape the acts as a protective barrier and prevents evaporation of reagents and helps to limit back-flow of reagents. When using some particularly delicate materials a cover tape is essential to maintain integrity of the test. The tape may have any number of designs imprinted upon it, such as test identification or trade names. For obvious reasons the cover tape must be clear over the test and control line sections of the ICS assay. As with the “Plastic-adhesive backing card”, discussed above, a fine balance must be achieved between adhesive strength and migration of the adhesives to prevent assay interference and loss of bed-volume.

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9. Strip housing/Cassette

A plastic housing typically made of two pieces that snap together and protect the assembly. The test strip and absorbent pad are contained within this housing that allows the unit to be handheld more easily and protects the strip from damage and environmental contamination. A window on the side of the housing allows the test and control zone portions of the strip to be viewed. In some instances this is little more than a hole in one half of  the cassette but it may also be a clear plastic window that protects the membrane from being accidentally damaged or splashed while still allowing visualization of the strip.

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