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Originally published May 1996

The use of microporous polymer membranes in immunoassays

Michael A. Harvey, Charlene A. Audette, and Richard McDonogh

Microporous membranes have proven to be highly successful surfaces for the development of simple, rapid immunoassay delivery systems. Membranes made of nitrocellulose, currently the material of choice, can be produced with a wide range of pore sizes and treated with surfactant to optimize their performance. These membranes help create sensitive, stable, and reproducible assays.

Since the first demonstration in 1979 that proteins could be transferred to microporous nitrocellulose membranes and detected using antibodies,¹ development of rapid immunoassays using these high-surface-area materials has proliferated.^2­7 Initially much investigation centered on understanding the interactions between proteins and polymers and the requirements for blocking nonspecific interactions on the membrane, and on developing a series of detection methodologies and strategies. This work has led to a variety of immunoassay delivery systems for detecting a large menu of analytes. Membranes are used in both sandwich and competitive immunoassays to detect both small and large molecules.^8,9

Key membrane characteristics include polymer type, porosity, surfactant content, and strength. Because the performance and capabilities of membrane-based immunoassay systems are influenced by specific properties of the membranes, a thorough characterization and understanding of these factors is essential.

Types of Membranes

The choice of a membrane for an immunoassay delivery system depends largely on three properties: protein-binding capacity, porosity, and strength. The ability of the membrane to immobilize macromolecules, in particular proteins, is paramount, since they are the solid phase used in the assay. The porosity of the membrane is important because reactants must flow through the matrix so that it can separate bound from free components. The strength of the membrane is important for the manufacture and eventual use of a device.

Polymers tested for their ability to bind proteins include cellulosics (nitrocellulose, cellulose acetate, regenerated cellulose), nylon, and polyvinylidene fluoride (PVDF), as shown in Figure 1. The binding capacities of cellulose acetate and regenerated cellulose are insufficient for most immunoassays, but a range of higher capacities are available with nitrocellulose, nylon, and PVDF membranes. (Figures not yet available on-line.)

Most current membrane-based immunoassays use either nylon or nitrocellulose. Both bind proteins noncovalently, although the mechanism of interaction is probably different. A polyamide, nylon binds proteins via electrostatic and charge interactions, while the interaction of nitrocellulose with proteins appears to be primarily hydrophobic in nature.¹⁰ Both polymers bind most proteins--in particular, antibodies--with sufficient density and avidity to support an immunoassay. They generally do not require covalent attachment of the immobilized reactant.

Although nylon was perhaps the first polymer membrane employed in a commercial immunoassay delivery system,¹¹ nitrocellulose has proven to be more versatile and is currently the membrane of choice. While the protein-binding capacity of nitrocellulose is lower than that of nylon, it is sufficient to support sensitive immunoassays. This allows nitrocellulose-based assays to avoid the high level of nonspecific interactions that can contribute to background in nylon-based assays. Nitrocellulose can be manufactured in a wide range of porosities, from 0.05 to about 12 µm, providing in turn a range of binding capacities and wicking or capillary-rise rates to aid in the flow of reactants through the assay.

Flow-Through and Lateral-Flow Immunoassays

In choosing a particular nitrocellulose membrane for an immunoassay system, the role the membrane plays in the assay must be considered. Membrane-based assays fall into two broad categories. Historically, the first such assays were flow-through filtration assays (Figure 2). In making this type of assay, one immunoreactant is immobilized to a defined area on a membrane surface. This membrane is then overlaid on an absorbent layer that acts as a reservoir to pump sample volume through the device. Following immobilization, the remainder of the protein-binding sites on the membrane are blocked to minimize nonspecific interactions. When the assay is used, a sample containing analyte is added to the membrane and filters through the matrix, allowing the analyte to bind to the immobilized antibody. In a second step, a tagged secondary antibody (an enzyme conjugate, an antibody coupled to a colored latex particle, or an antibody incorporated into a colored colloid) is added that reacts with captured analyte to complete the sandwich. Alternatively, the secondary antibody can be mixed with the sample and added in a single step. If analyte is present, a colored spot develops on the surface of the membrane. This type of assay has the advantage of simplicity both in manufacturing and in use.

The choice of a membrane for such an assay depends on several factors, such as the sensitivity of the assay, the time required to run the assay, and the type of readout employed in the assay. The pore size of the membrane is the key factor. Nitrocellulose membranes with pore sizes from 0.2 to 8 µm have been used in flow-through assays. The larger the pore size, the lower the surface area and, therefore, the lower the protein-binding capacity. But because membranes with larger pores support more rapid filtration rates, they can yield a faster assay. Conversely, a smaller-pore membrane can create a more sensitive assay.

The size of the tagged antibody complex may also dictate the pore size, which must be large enough to permit unreacted antibody to pass through. Assays that use an antibody coupled to a colored latex bead require a larger pore size.

More recently, lateral-flow immunoassay systems have been developed to allow for single-step assays that require only the addition of a sample (Figure 3). In these assays, the sample is added to one end of the device and flows by capillary action through the interstitial space of the materials in the device. While continuing along this flow path, the sample contacts dried reagents, usually tagged secondary antibodies, which then migrate with the analyte to a capture zone of immobilized antibody on the membrane. Unreacted tagged antibody continues to flow past this capture zone, normally to an end-of-assay indicator. Generally, absorbent material at the distal end of these devices helps draw the sample through the device.

Membranes in these types of systems must offer different properties than those best used in flow-through devices. Capillary rate becomes a more important property, since it not only dictates the total time required to run the assay but also determines the residence or reaction time at any given zone.

To accommodate these requirements, nitrocellulose membranes with larger pore sizes, up to 12 µm, have been developed. As the pore size increases, the speed of wicking or capillary rise through the matrices increases (Figure 4). However, the relationship between wicking rates and distance is not linear (Figure 5); thus reaction time at any point along a membrane may vary. Even though there is a considerable decline in the binding capacity of a large-pore-size nitrocellulose membrane, it retains sufficient capacity to support all immunoassays (Figure 6).

Pore size is not the only factor that can affect the capillary rate. Most nitrocellulose membranes contain a surfactant to aid in wetting and act as an antistatic agent. The amount and type of surfactant included in a membrane can be altered to impart different capillary rates to membranes with equivalent pore sizes (Figure 7).

Manufacturing Considerations

Tensile Strength of Nitrocellulose Membranes. A property of nitrocellulose that challenges its use in immunoassay formats is its low tensile strength. Three different solutions to providing sufficient strength have been used.

Some nitrocellulose membranes can be cast around a supporting, presumably noninteractive, material such as a nonwoven polyester. The most common approach, however, has been to laminate the membrane to a plastic backing with an adhesive. A third approach, intended to eliminate the potentially detrimental interactions between membrane and adhesive, has been to cast the nitrocellulose membrane directly on a plastic support. This eliminates the need for a lamination step in manufacturing as well as the problems that can be encountered with the use of adhesive systems.

Storage of Nitrocellulose Membranes. In order for nitrocellulose membranes to preserve their protein- interactive properties, they must be properly stored, to remain hydrated. Dried membranes will not rehydrate easily, and will become more brittle and perhaps lose binding capacity. Prior to device manufacturing, nitrocellulose membranes should be stored in an environment of controlled humidity (40­60%) and controlled temperature (18°­20°C).

Application of Reagents to a Nitrocellulose Membrane. In order to establish a reproducible, sensitive assay on a membrane, the various immunoreactants must be accurately applied to the membrane. Generally, antibodies or antigens are added as a line or a symbol covering a defined surface area. Most antibody lines have a width of 1­2 mm. The volume required to produce such a line is in the submicroliter range, with application rates between 0.5 and 1.0 µl/cm of membrane.

Instrumentation to attain such rates must be reproducible and accurate. One application method is airbrushing, which involves moving the membrane past an airbrush head at a controllable rate. A second and perhaps preferred method is to use a positive-displacement micropipetting system. Manufacturers that provide such systems include Ivek Corp. (North Springfield, VT), BioDot (Irvine, CA), and Ismeca USA (Carlsbad, CA).

Additional Treatments of the Membrane. Following application of the immunoreactants to the membrane, further treatment of the latter falls into two categories. One treatment sometimes required is a drying or curing step to maintain the immunoreactivity of reagents and to fix reactants to the membrane. This is often accomplished by desiccating the membrane, sometimes at an elevated temperature.

The second postapplication treatment sometimes employed is to block the membrane to prevent subsequent nonspecific interactions. A variety of membrane blockers have been successfully employed, most commonly proteins. The membrane is typically dipped in a high concentration of a protein, such as albumin, and allowed to dry. Other systems instead incorporate a blocker into a release pad to pass through the membrane with the sample. Surfactants are often used for this purpose. One problem that may be encountered when blocking a membrane with a protein is a significant decline in capillary rate.

Conclusion

Characterization and modification of the physical properties of microporous membranes to facilitate their use in immunoassay delivery systems has led to the development of a new generation of nitrocellulose membranes. These membranes are cast on different supports and have modified surfactant content and a range of pore sizes. They are currently used as components of human and veterinary diagnostics and food tests and are making their way into new measurement technologies in environmental and agricultural testing. They will also play a role in molecular-biology-based (genetic) assays.

References

1. Towbin H, Staehelin T, and Gordon J, "Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications," Proceedings of the National Academy of Sciences of the United States of America, 76:4350­5354, 1979.

2. Rudolph JL, Henderson MB, Chow O, et al., "A New Microporous Membrane for Diagnostic Immunoassays," Biotechniques, 9:218­220, 222­223, 1990.

3. Snowen K, and Hommel M, "Antigen Detection Immunoassay Using Dipsticks and Colloidal Dyes," J Immunol Methods, 140:57­66, 1991.

4. Buechler KF, Moi S, Noar B, et al., "Simultaneous Detection of Seven Drugs of Abuse by the Triage Panel for Drugs of Abuse," Clin Chem, 38:1678­1684, 1992.

5. Van Amerongen A, Wichers JH, Berendsen L, et al., "Colloidal Carbon Particles as a New Label for Rapid Immunochemical Test," J Biotechnol, 30:185­195, 1993.

6. Rao DV, and Kashyap VK, "A Simple Dipstick Immunoassay for Detection of A and B Antigens," J Immunoassay, 13: 15­30, 1992.

7. Liu H, Yu JC, Bindra DS, et al., "Flow Injection Solid Phase Chemiluminescent Immunoassay Using a Membrane-Based Reactor," Anal Chem, 63:666­669, 1991.

8. Kudo T, Iqbal K, Ravid R, et al., "Ubiquitin in Cerebrospinal Fluid: A Rapid Competitive Enzyme-Linked Immunoflow Assay," Neuroreport, 5:1522­1524, 1994.

9. Sonnenberg A, Kolvenbag G, Al E, et al., "Immunobinding Procedure with Monoclonal Antibodies," J Immunol Methods, 72:443­450, 1984.

10. Gershoni JM, and Palade GE, "Electrophoretic Transfer of Proteins from Sodium Dodecyl Sulfate Polyacrylamide Gels to a Positively Charged Membrane Filter," Anal Biochem, 124:396­405, 1982.

11. Levinson PA, Owen CN, and Valkirs GA, Method and apparatus for immunoassays: binding enzyme-labeled antibody to antigen on porous membrane, U.S. Pat. 4,632,901.

Michael A. Harvey, PhD, is vice president, research and development, and Charlene A. Audette is a research associate at Schleicher and Schuell, Inc. (Keene, NH). Richard McDonogh, PhD, is director of research and development, Schleicher and Schuell GmbH (Dassel, Germany).