IVD Lateral Flow – Sample, Conjugate, and Absorbent Pad Basics

Sample, Conjugate, and Absorbent Pad Materials

Lateral flow pad materials comprise the porous matrices that are used for the sample pad, conjugate pad, and absorbent pad. Most commonly, cellulosic materials (i.e., filter papers) are used for sample and absorbent pads, while glass fiber filters are used for the conjugate pad. We offer a range of cellulosic and glass fiber materials under the SureWick^® trademark (Table 1). These materials are typically less costly than nitrocellulose membranes as they are easier to manufacture. In most cases, however, these materials are not being manufactured specifically for use in lateral-flow tests.

Depending on where pad materials are sourced, they may lack specifications that pertain to lateral-flow tests and exhibit levels of variability that are greater than desired, particularly at the dimensions that they are being used in lateral-flow tests. Other materials, such as woven meshes and synthetic nonwovens, are also used as pad materials, although at a much lower frequency.

• Sample Pad Selection and Specifications
  • Specifying the Sample Pad
• Conjugate Pad Selection and Specifications
  • Specifying the Conjugate Pad
• Absorbent Pad Selection and Specifications
  • Specifying the Absorbent Pad
  • Manufacturing Plan in Each Phase of Development

Chemistries on SureWick^® Pad Materials

The sample and conjugate pad serve as the location for chemicals that are essential to the running of the lateral flow test. Specific details are described below for each type of pad. There is one important aspect of these chemicals that is generally unappreciated. The chemicals are loaded into these pads by applying homogenous solutions and evaporating the water. The solutes may become heterogeneous during the evaporation process because of different solubilities. Liquid constituents, such as TWEEN^® 20, will return to a highly concentrated state and probably are not evenly distributed across the fibrous network of the pad. When sample is applied to the test strip, their rates of dissolution will depend on their inherent solubilities and the rate of mixing at the molecular interface. It should be expected that these constituents will not resolubilize at the same rates when the sample is applied to the test strip. Thus, more rapidly solubilizing constituents will be more concentrated near the liquid front, while those that solubilize more slowly will be delayed in their release into the liquid stream. The net effect of these phenomena is to generate a set of chemical gradients within the liquid stream as it moves through the pads and onto the membrane.

There may be locations within the liquid stream where the concentration of the chemicals is not conducive to interaction between the antibodies and analyte.

Table 1 SureWick^® Pad Materials

Intended Use	Material	Code	Thickness (mm)
Sample/absorbent pad	Cellulose	C048	0.48
Sample/absorbent pad	Cellulose	C068	0.68
Sample/absorbent pad	Cellulose	C083	0.83
Sample/absorbent pad	Cellulose	C248	2.48
Conjugate Pad	Glass Fiber	G028	0.28
Conjugate Pad	Glass Fiber	GFDX	0.43

Sample Pad Selection and Specifications

The sample pad (Figure 1) can be used to perform multiple tasks, foremost of which is to promote the even and controlled distribution of the sample onto the conjugate pad. It may also control the rate at which liquid enters the conjugate pad, preventing flooding of the device. When impregnated with components such as proteins, detergents, viscosity enhancers, and buffer salts, the sample pad can also be used to:

1. Increase sample viscosity (modulate flow properties).
2. Enhance the ability of the sample to solubilize the detector reagent.
3. Prevent the conjugate and analyte from binding non-specifically to any of the downstream materials.
4. Modify the chemical nature of the sample so that it is compatible with immunocomplex formation at the test line.
5. Promote even flow of the sample along the membrane.

The presence of added protein (such as albumin) and detergents and surfactants (such as SDS or TWEEN^® 20 at a low concentration) may promote resolubilization of the conjugate, reduce nonspecific binding of the conjugate, and possibly minimize adsorption of the analyte to the membrane.

Figure 1. Sample Pad

By adding blocking agents to the sample pad, it may be possible to eliminate blocking of the membrane. This approach may be much easier and considerably more cost-effective than attempting to block the membrane directly. Unless the antibody (or antigen) is covalently attached to the detector particle, it is not advisable to dry the detector reagent into the conjugate pad in the presence of blocking proteins or detergents. Exogenous proteins, especially in the presence of detergents, can displace the antibody or the antigen from the detector particle during prolonged storage. Thus, the sample pad may be the only place in the test device other than the membrane where blocking and resolubilization agents can be added safely.

Some tests require samples that exhibit wide variation in chemical composition. Human urine, for instance, can have a pH between 5 and 10. Differences in pH and ionic strength may shift the specificity and sensitivity of capture and detector reagents and promote varying degrees of non-specific binding of detector reagents due to changes in charge densities. Adding a relatively high concentration of buffer salts to the sample pad (for example, by pre-treating with 1.0 M borate buffer, pH 9.5) can minimize variation by controlling the pH and ionic strength of the solution that emerges from the sample pad.

There are two types of materials that are commonly used as sample pads: cellulose fiber filters and woven meshes.

Woven meshes, sometimes called screens, normally work very well to distribute the sample volume evenly over the conjugate pad. They also typically have good tensile strength and handle well, even when wet. Meshes have very low bed volumes, meaning that they retain very little sample volume, normally 1 – 2 μL/ cm². On the other hand, it is impractical to treat them with the intention of loading them with enough solutes to modify the protein content, pH, ionic strength, or viscosity of the test sample. Meshes can also be expensive relative to other porous media and difficult to process through strip cutting machinery.

Cellulose filters have properties that are nearly the opposite of woven meshes. They are thick (> 250 μm), weak, and relatively inexpensive. Cellulose filters also have large bed volumes (> 25 μL/ cm2). Paper can be very difficult to handle, especially when wet. Cellulosic filters are the most commonly used materials to make the sample pad because they can be loaded with a wide array of blocking agents, detector reagent release agents, pH and ionic strength modifiers, and viscosity enhancers. When using cellulose filters, care must be taken to ensure sufficient and consistent contact with the underlying conjugate pad material. Failure to achieve good contact and adequate compression can lead to interrupted or inconsistent transfer of fluid into the conjugate pad.

For many urine-based assays, especially pregnancy and ovulation tests, porous plastic wicks protrude from the end of the cassette. Their primary function is to collect liquid from the urine stream so that it can be transferred to the test strip within the cassette. The dimensions of the wick are tailored to the design of the test strip cassette and the requirement to absorb enough liquid to run the test. The wick may or may not be chemically treated, depending on the chemistries of the other materials within the cassette. It is conceivable that the plastic wick can serve the function of a sample pad.

Specifying the Sample Pad

Specification of the sample pad depends greatly on its intended purpose in the test device. If the sample pad is being used primarily to modify the sample, the following attributes should be specified:

Thickness (Mean and Variability)

Mean thickness may be given as microns, millimeters, or thousandths of an inch (mils). The range of variability is also critical since this will affect the amount of bed volume and the consistency of compression in a housing. For strips placed in housings, the sample is typically applied to a port that exposes a small region of the sample pad. If the pad material is too thick, the fibers may be compressed so that absorption of liquid into the pad is greatly reduced or prevented. If the pad material is too thin, there may be little or no contact with the housing. This allows the sample to enter the housing unobstructed, flooding the interior, and significantly altering the flow dynamics of the test strip.

Basis Weight (Mean and Variability)

The basis weight is the mass of fibers per unit area. In the paper and non-woven industries, it is most often expressed as g/cm². This value is of little relevance for lateral flow tests. Using the basis weight, the thickness of the material, and the density of the polymer, the bed volume and porosity can be calculated. Bed volume is directly proportional to thickness at constant porosity. Similarly, bed volume is directly proportional to porosity at constant thickness. Thus, variability in basis weight can be equated to variability in bed volume. Bed volume is actually the critical performance parameter, but it is rarely provided.

Tensile Strength

Tensile strength for sample pad materials is important for the same reason as for membranes. Some materials are as weak as unbacked membranes. Since they may be slit to widths of 1 cm or less, web handling can be very difficult in a continuous processing operation.

Extractables

Materials used to make sample pads contain binders to hold the fibers together. In addition, some of the fibers may break or not be interconnected with the pad’s macrostructure. Consequently, a considerable percentage of the pad’s components may be dislodged during various processing steps. When a test strip is run, this can lead to plugging and poor fluid transfer as the sample wets out the pad and moves downstream.

Dimensions (Length, Width)

The dimensions and tolerances should be completely defined.

Packaging, Labeling, and Certification

The considerations pertaining to membranes also apply to sample pad materials.

Particle Retention Rating

In some applications, the sample pad is used as a filter to remove particles from the sample before the liquid enters the conjugate pad. Thus, it is important to know the particle retention rating. Since these materials are depth filters, they do not exhibit 100% retention capability. Because of the way that papers and non-woven materials are manufactured, changing the particle retention rating often involves significant changes to the thickness and basis weight.

Conjugate Pad Selection and Specifications

The conjugate pad (Figure 2) can perform multiple tasks, the most important of which is uniform transfer of the detector reagent and test sample onto the membrane. When sample flows into the conjugate pad, the detector reagent solubilizes, lifts off the pad material, and moves with the sample front into the membrane. The ideal conjugate pad material has the following attributes:

1. Low non-specific binding: If the detector reagent or analyte binds to the conjugate pad, it will be unavailable to form the immunocomplex at the test line, thereby reducing signal intensity and sensitivity.
2. Consistent flow characteristics: Inconsistent flow properties can cause serious performance problems. If the material does not deposit the sample uniformly onto the membrane, the detector reagent may be channeled onto the membrane, appearing as streaks as the sample migrates along the membrane. Consequently, there will be uneven signal development at the test and control lines.
3. Consistent bed volume: When loaded into the conjugate pad by dipping, the amount of detector reagent in each test strip depends on the bed volume of the material. If the bed volume varies significantly, variable signal intensities may be observed even though all other components of the strip are constant.
4. Low extractables: In addition to chemical extractables, the material should be free of particles that can clog the membrane at the conjugate pad/membrane interface.
5. Good web handling characteristics and consistent compressibility: These attributes are important for incorporation into test strip manufacture and for consistent liquid transfer onto the membrane.

Figure 2. Conjugate pad for lateral flow assays.

The Role of the Conjugate Pad in Assay Sensitivity

An important function of the conjugate pad is to deliver the detector particles onto the membrane in a consistent volume of sample on every test strip. Ultimately, the sample volume required to release the detector particle into the sample stream determines how much analyte can be measured. Only the analyte contained in the volume of sample that migrates ahead of and with the detector particles can contribute to the signal. The volume of sample that enters the conjugate pad and membrane after the detector particles have been completely released does not contribute to signal, although it does serve to reduce assay background (Figure 3).  Analyte that passes over the capture reagent line after all of the detector particles have migrated farther downstream may bind at the capture reagent line but will lack additional detector particles to complete the immunocomplex. The sample volume actually analyzed in the test strip equals the amount of sample required to solubilize the detector particles, not the total amount absorbed by the device.

Figure 3. Lateral flow assay sensitivity.

The porous materials commonly used for conjugate pads are non-woven filters, which are manufactured by compressing fibers of cellulose, glass, or plastic (such as polyester, polypropylene, or polyethylene) into thin mats. They are specified by fiber size, thickness, basis weight, extractables, and air flow rate. In most cases, they cost considerably less than membranes. Materials commonly used to make conjugate pads include glass fiber filters, cellulose filters, and surface-treated (hydrophilic) polyester or polypropylene filters. The key properties for each are summarized in Table 2.

Table 2 Properties of conjugate pad materials

Non-woven Material	Brief Description	Key Benefits	Key Liabilities
Glass fibers	100 – 500 μm thick, may contain binders to hold fibers together	Good hold-up volumes, low nonspecific binding	Poor tensile properties, difficult to slit and web handle
Cellulose filters	300 – 1000 μm thick, compact fibers of consistent density	Very low nonspecific binding, normally very uniform	High hold-up volumes (> 50 μL/ cm2), can be very weak when wet
Surface-modified polyester	100 – 300 μm thick, hydrophilic polyester filters	Low nonspecific binding, excellent tensile strength and web handling	Low, and somewhat variable, hold-up volumes (< 15 μL/ cm2)

Specifying the Conjugate Pad

The conjugate pad is as critical as the membrane in controlling the performance of lateral flow tests. As such, it is very important to define key material specifications:

Thickness (Mean and Variability)

Thickness may be given as microns, millimeters, or thousandths of an inch (mils). The range of variability is also critical since this will affect the bed volume and the consistency of compression in a housing.

Basis Weight (Mean and Variability)

The basis weight is the mass of fibers per unit area. In the paper and non-woven industries, it is most often expressed as g/m². This value is of little relevance for lateral flow tests. Using the thickness of the material and the density of the polymer, the bed volume and porosity can be calculated. Bed volume is directly proportional to thickness at constant porosity. Similarly, bed volume is directly proportional to porosity at constant thickness. Thus, variability in basis weight can be equated to variability in bed volume. Bed volume is actually the critical performance parameter, but it is rarely provided.

Tensile Strength

Tensile strength for conjugate pad materials is important for the same reason as for membranes. Some materials are as weak as unbacked membranes. Since they may be slit to widths < 1 cm, web handling can be very difficult in a continuous processing operation.

Extractables

Materials used to make conjugate pads may contain binders to hold the fibers together. In addition, some of the fibers may break or not be interconnected with the pad’s macrostructure. Consequently, a considerable percentage of the pad’s components may be dislodged during various processing steps. When a test strip is run, this can lead to plugging and poor fluid transfer as the sample wets out the pad and moves downstream. Extraneous glass fibers can also present a health hazard in automated manufacturing systems.

Dimensions (Length, Width)

The dimensions and tolerances should be completely defined.

Packaging, Labeling, and Certification

The considerations pertaining to membranes also apply to conjugate pad materials.

Absorbent Pad Selection and Specifications

Selecting an Absorbent Pad

Absorbent pads, when used, are placed at the distal end of the test strip (Figure 4). The primary function of the absorbent pad is to increase the total volume of sample that enters the test strip. This increased volume can be used to wash unbound detector particles away from the test and control lines, thereby lowering the background and enhancing assay sensitivity. Since the volume of sample that ultimately contributes to signal is controlled by the volume required to solubilize the detector particles, and not by the total volume of sample that enters the device, the addition of the absorbent pad may not have a dramatic impact on overall assay sensitivity. If the strip design does not include an absorbent pad, the volume of sample analyzed in the strip is determined solely by the bed volume of the membrane.

Figure 4. Absorbent pad for lateral flow.

There are two major considerations associated with the use of absorbent pads. First, a suitable material must be identified, specified, purchased, and integrated into the manufacturing process. Ultimately, this leads to a higher cost for the finished product. Second, an absorbent pad makes it difficult to incorporate an end-of-assay indicator in the test device. The flow of liquid through the absorbent pad is not necessarily laminar, and the pad may fill with liquid unpredictably.

Most absorbent pads are made from cellulose filters. The material should be selected on the basis of thickness, compressibility, manufacturability, and, most of all, uniformity of bed volume. Once an absorbent material has been chosen, optimizing the overall volume absorbed by the test strip is best managed by changing the dimensions (usually the length) of the absorbent pad.

Specifying the Absorbent Pad

All of the specifications for the sample pad apply to the absorbent pad, with the exception of extractables.

Consider the Manufacturing Plan in Each Phase of Development

Manufacturing schemes range from entirely manual to completely automated. For reproducibility, there are certain steps that require a high level of consistency.

Choose the Right Manufacturing Process

The choice of process depends primarily on the amount of product to be manufactured and the funds available to staff and equip the manufacturing facility.

• Manual – process materials in a batch mode.
• Automated – process materials in a continuous mode.
• Combination of manual and automated.

Key Elements for Manufacturing Consistency

Consider the following elements of the manufacturing process to ensure consistency from R&D through manufacturing.

• Consistent lamination of membranes, sample pads, conjugate pads, and absorbent pads onto a support backing.
• Precision cutting of sheets or rolls into strips of defined length and width.
• Control for consistency of reagent application onto membranes, sample pads, conjugate pads, and other porous media.
• Placement of test strips in plastic housings.

Minimize Waste, Maximize Yield

And finally, when designing the assay and planning for progression to a manufactured product, use the following steps to minimize waste and maximize yield.

• Match lot sizes of materials and reagents during planning.
• Continuously train personnel to ensure proper execution of manufacturing processes.
• Continuously conduct in-process inspections to ensure sub-assemblies are being manufactured to specification.
• Ensure that materials chosen are compatible with manufacturing machinery so they perform predictably in the finished device.