Originally Published IVD Technology January 2002
Qualification of cellulose nitrate membranes for lateral-flow assays
Ensuring the suitability of membranes involves numerous factors.
Hans H. Beer,
Eric Jallerat, Karl Pflanz, and Timo M. Klewitz
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Figure
1. A membrane winding at the end of a casting machine.
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Microporous cellulose
nitrate membranes are the oldest synthetic membranes and have a long and successful
history in many filtration applications. Eventually, their high but undesirable
binding capacity somewhat restricted their use in filtration applications. However,
these same characteristics provided opportunities for use in new types of applications
based on the immobilization of proteins, including blotting techniques, enzyme-linked
immunosorbent assay (ELISA) testing, and lateral-flow immunochromatographic
tests.
For such applications,
features such as migration speed, binding capacity, and absorption time have
become typical parameters of a diagnostic membrane’s specification. By
themselves, however, these features have proved insufficient for a complete
characterization. For adequate qualification that comes closest to actual practice,
dot and line tests with proteins are indispensable.
This article explores the challenges that are encountered by membrane manufacturers and the properties that are essential to qualifying membranes for IVD applications.
A Short History
Experiments for
the first generation of synthetic microporous membranes were performed by Wilhelm
Schumacher, using a special evaporation process on solutions of cellulose nitrate
in ether and alcohol. The fundamentals of a laboratory-sized preparation of
such membranes were published between 1907 and 1912, and were followed by systematic
developments toward economic industrial production of synthetic membranes from
cellulose acetate and cellulose nitrate. The first true industrial production
of membranes based on this advanced technology was started in 1929 at Sartorius
(Goettingen, Germany). Membrane production in the United States was initiated
at the California Institute of Technology (Pasadena, CA), supported by the experience
and patents of Richard Zsigmondy, who won the Nobel prize in chemistry in 1925.
At the beginning of the 1950s, the resulting process technology was transferred
to Lovell Chemical Co., which today has become Millipore Corp. (Bedford, MA).
Until the 1950s,
the major uses for such microporous cellulose nitrate membranes were filtration
applications that were based on the membranes’ ability to retain microorganisms
and particles from fluids, especially for the purpose of sterilization and microbiological
testing of liquids and air. With the large-scale application of sterile filtration
by means of pleated cartridges, it became essential to develop membranes with
a high mechanical strength and a low nonspecific binding of product by adsorptive
forces. Since cellulose nitrate by nature exhibits a unique, extremely high
binding capacity and rather poor mechanical strength, it was gradually replaced
by low-binding cellulose acetate and modified synthetic polymers, such as polyethersulfone
or polyvinylidene fluoride. As a result, its importance as a filtration material
became more or less restricted to microbiological testing, such as the identification
and quantification of microorganisms.
The high binding
capacity of microporous cellulose nitrate membranes for nucleic acids and proteins
depends on the high chemical affinity of the cellulose nitrate polymer for these
molecules, combined with the high void volume of microporous membranes, which
offers good accessibility and a large internal surface for the potential adsorption
of molecules. The binding of protein molecules to cellulose nitrate occurs almost
immediately, and although the actual mechanism is not yet completely understood,
it seems to depend mainly on hydrophobic and electrostatic interactions that
are caused by the nitrate dipole.1–5
The first important
application that was based on this unique binding capacity of cellulose nitrate
was membrane hybridization, using such membranes for immobilization and highly
specific detection of nucleic acid sequences by hybridization.6–8 Subsequently,
advanced blotting techniques combined with electrophoresis and related solid-phase
assays like ELISA and affinity chromatography followed.9–10 Most of these
applications of cellulose nitrate membranes remained confined to research labs.
The commercial-scale use of cellulose nitrate membranes for immobilization purposes
started at the end of the 1980s with the introduction of flow-through and lateral-flow
immunochromatographic tests based on antibodies, which are commonly known as
rapid IVD tests.11–14 At this point, the ability of cellulose nitrate to
form membranes of large pore size, up to about 15 µm, became another advantage,
making high capillary speeds possible.
Quality Issues
The use of cellulose nitrate membranes in immunochromatographic tests—and the optimization of the various components and reagents used with the membranes—have been discussed extensively in this and other publications. Despite all of this existing experience, diagnostic kit manufacturers are still facing large deviations in the test results, primarily in the shape and intensity of the test and control lines. In addition to the common variations of the different test components, these deviations may also be attributed to lot-to-lot inconsistency of the membrane. Diagnostic kit manufacturers need to avoid membrane-related variations at all costs. As a result, some of these manufacturers order membrane material only from a defined batch that has been pretested and proven to function correctly. Typical variations or membrane flaws affecting diagnostic applications include the following.
- Deviations in membrane structure (pore size or morphology, asymmetric structure on the top and bottom).
- Uneven wetting properties (inhomogeneous distribution of wetting agent in the matrix).
- Imperfections in surface quality (membrane dust, local occurrence of lines or spots).
Manufacturing Process. All cellulose nitrate membranes are made from solutions by a method that is known as evaporation or air casting (see Figure 1). A typical casting solution contains defined amounts of cellulose nitrate polymers, volatile solvents, cosolvents, nonsolvents of gradual volatility, and additives such as surfactants.
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Figure
2. Schematic of an evaporation casting line for manufacturing cellulose
nitrate membranes.
(click to enlarge) |
This solution is
applied with a defined film thickness onto a long, moving stainless-steel belt.
As the solvents vaporize, a solid microporous membrane layer is gradually formed
by precipitation of the polymer, which is then dried (see Figure 2). This entire
evaporation process requires precise conditions and proper in-process controls,
which can sometimes be difficult to obtain with the usually large-sized industrial
equipment.
Design and quality
of the casting equipment are also critical and often responsible for some types
of variation.
Raw Material. All types of cellulose nitrate polymers that are used in a common casting solution originate from natural sources, such as cotton or wood pulp, and are thus subject to certain regional and ‘vintage’ variations. These variations codetermine the distribution of polymer chain length, which is an essential factor in the ability of the raw materials to form membranes. Of course, the individual production processes of the suppliers will also affect the quality of the raw materials.
Characterization
and Qualification
Membrane manufacturers
have continuously tried to reduce the range of lot-to-lot membrane variation
with varying levels of success. These manufacturers have implemented several
in-process controls and specific final tests for better qualification and selection
of membrane lots for diagnostic applications. The following standard properties
have proven to be essential for diagnostic applications and are currently most
often used for membrane specification.
Lateral Wicking
Speed. Originally used in the paper industry for the characterization of
porous papers, this test was modified and first used by a diagnostic kit manufacturer
before it was adopted by membrane suppliers.
This test is simple and highly informative. The standard today is to measure the wicking time for a distance of 40 mm of membrane in a vertical position. The commonly used test medium is water, but aqueous buffers containing dyes are also employed, yielding somewhat longer times than pure water. Since the results also depend on the temperature of the test liquid and the moisture content of the membrane, defined test conditions have to be observed and specified by the membrane manufacturer.
Wicking time is
generally measured across the membrane roll, which corresponds to the flow direction
of the final test strip and is given in units of second per 40 mm. This number
represents the fundamental parameter of a diagnostic membrane and is mostly
used by membrane manufacturers to define different types of membranes. Since
membrane casting is a directed process with a preferential orientation, migration
along the roll always exhibits a faster speed.
This test provides
a combined evaluation of the lateral pore size structure and wettability, such
as the activity of the wetting agent. Additionally, the shape of the flow front
provides information about surface quality and homogeneity of the membrane as
well as the dispersion of the wetting agent within the membrane.
Today, manufacturers
can maintain and guarantee a lot-to-lot variation of less than ±25% for
the nominal capillary speed of a particular membrane. This number expresses
the maximum variation observed in sample measurements from all the different
membrane batches released, and thus encompasses both inter- and intralot variation.
However, intralot variation by itself is generally much smaller, in the range
of ±10% or less. When expressing their range of variation as a standard
deviation, manufacturers commonly apply the ±3 sigma rule; thus a total
maximum variation of 25% would yield a standard deviation of 8.33.
Thickness. This
parameter is easy to measure and seems rather trivial. However, it is a critical
factor for both membrane production and membrane application. Together with
the casting formula and the climatic conditions, the thickness of the cast film
influences the evaporation and diffusion process during membrane production
and determines such fundamental properties of the final solid membrane as pore
size and structure.
These properties
are relevant to application parameters like flow rate, migration speed, and
binding capacity. In combination with morphology, wettability, and binding capacity,
membrane thickness can affect the resulting width of protein lines that are
applied to a membrane surface as well as the final distribution of bound protein
that is inside the membrane matrix. These individual factors are important,
especially if a quantitative evaluation of the resulting signals by means of
light reflection or transmission is intended.
Absorption Time and Absorption Capacity for Liquids. This test evaluates the time required for a membrane to take up a certain volume of liquid medium, and the area of the resulting wet spot, which directly depends on void volume and thickness. These parameters are usually called absorption time and absorption capacity, and are respectively reported in seconds per µl and cm2/µl.
Besides the viscosity and surface tension of the test medium, the absorption time depends on pore size, structural quality, and the hydrophilic properties of the membrane, such as its moisture and wetting-agent content. Therefore, exact climatic conditions have to be maintained with this test.
Total Protein Binding Capacity. This term is used by several membrane manufacturers to express the total amount of protein that a membrane is able to bind through adsorption. This test method requires covering the entire membrane with a monolayer of protein by shaking in an excess amount of immunoglobulin G (IgG) or albumin solution, removing the excess protein by washing with a buffer, desorbing the membrane-bound protein with a strong surfactant, staining the resulting solution with a protein dye, and photometrically evaluating the total amount of protein.
The binding capacity
of the membrane is commonly specified in an area-related unit, such as µg/cm2,
or a volume-related unit, such as µg/mm3. Binding capacity depends primarily
on the pore size of the membrane or, more specifically, the total internal surface,
which increases as the pore size decreases. According to measurements using
the Brunner Emmet Teller (BET) method, typical diagnostic membranes with a nominal
pore size of 3 to 8 µm exhibit a surface area ratio of 40 to 20 m2 per
gram of membrane layer, which is equivalent to around 0.5 to 1.0 m2 per frontal
cm2 of membrane.
Surfactants are always used in cellulose nitrate casting solutions for adequate wetting of the dry membrane. These generally affect the binding capacity and should therefore be used only at the minimum concentration required.
Specific Protein
Dot and Line Tests
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Figure
3. Dispensed lines of different molar concentrations of BSA (a) and IgG
(b). In each example, the top line has a concentration of 1.0 5 10–5
mol/L, the middle line is 6.7 5 10–6 mol/L, and the bottom line is
3.3 5 10–6 mol/L. All of the lines are stained with Ponceau. The
BSA lines are wider than with IgG, due to different diffusion and binding
mechanisms.
(click to enlarge) |
The binding capacity of a membrane and the corresponding coverage rate also depend on the binding mechanism and the properties of the test protein, such as its spatial structure, the type and number of binding sites, and electrical charge.15 For example, although IgG molecules are much bigger than albumin molecules, cellulose nitrate membranes bind the same molar amounts per volume unit—that is, about twice the weight of IgG compared with albumin. These relations can be directly demonstrated by dot or line tests with different protein solutions of equimolar concentrations, resulting in corresponding dot sizes or line widths on the same membrane (see Figure 3). In fact, the total binding capacity test is now being replaced or supplemented by these more-informative dot and line tests, in which only small areas of the membrane are covered with protein, as is done in actual applications.
Despite the increasing precision of physical membrane parameters, diagnostic kit manufacturers may still find batch-to-batch differences at various manufacturing steps. The first step is dispensing reagents, usually antibodies, onto the membrane to prepare the capture and control lines. At this stage, differences in wetting time (the time necessary for the reagents to be completely taken up by the membrane) or in the width of the applied lines, may become apparent.
The second step
is running the final assay under standard conditions. At this point, lines of
different intensity and width may result. If other causes can be excluded, these
variations can arise only from variations in membrane quality, such as pore
size, morphology, surfactant concentration, or surface quality. In particular,
a secondary unwelcome phenomenon during membrane formation is the presence of
varying quantities of membrane dust, which results from raw material variations
and the individual production process.
To assess these
differences, which cannot be detected using the standard physical tests described
above, membrane manufacturers should implement tests that utilize all of the
interactions of a membrane during the application of the protein lines as well
as during final performance of an immunochromatographic assay. These tests can
only be attained with the same dispensing equipment, reagents, and conditions
as those that are utilized by kit manufacturers.
A variety of protein
tests are already being used to some extent as an incoming quality control inspection
by some kit manufacturers. These dot and line tests are more informative than
other test methods, since they are closer to practice. Since protein concentrations
are always used in practice far below the binding capacity of the membrane,
the final spatial extension of dyed protein is always smaller than the original
wet reagent lines or spots.
Dot Tests. The dot tests involve the precise application of defined volumes of buffered protein solutions, such as albumin or IgG, at different concentrations onto dry membrane. The colorimetric determination is made using stained proteins or proteins treated with a subsequent staining and destaining process similar to that used for serum protein electrophoresis on cellulose acetate membranes.
Line Tests. The line tests entail the application of protein lines onto a dry membrane by means of dispensing equipment such as that used in the production of diagnostic kits. The exact conditions of adequate dispensing rate and speed have to be optimized with respect to membrane characteristics such as pore size, binding capacity, and uptake properties, as well as protein solution characteristics such as viscosity and surface tension.
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Figure
4. The symmetrical density profile of a BSA line on a regular membrane
indicates uniform binding of the BSA to the membrane (blue). The asymmetrical
density profile of a BSA line on an irregular membrane indicates nonuniform
binding of the BSA to the membrane (red).
(click to enlarge) |
The operating mode of the dispensing equipment will determine the initial contact pattern and the penetration depth of liquid into the membrane. The design of the membrane has an impact on the wetting shape and the progress of liquid flow inside the membrane. With a supported membrane, the air that is contained in the pores is only able to escape laterally, whereas it will escape backwards on an unsupported membrane. The same methods as those used for dot tests can be applied to make the bound protein visible.
Protein lines stained with Ponceau S and obtained from two batches of a membrane having the same characteristics according to standard physical tests are very different with regard to line formation, especially when using albumin as a test protein. However, the optimal technique is to use antibodies for the preparation of the lines and to make them visible by migrating corresponding target proteins that are conjugated to gold or dyed latex particles according to the principle of immunochromatographic tests. With this technique, the migration time can also be measured.
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Figure
5. A protein line pattern on a regular membrane (a) and an irregular membrane
(b). In each example, the top line is IgG and the bottom line is BSA.
(click to enlarge) |
Evaluation. The size, shape, and intensity of the bound protein dots or lines are usually evaluated by visual inspection. For a more precise differentiation of individual signals, quantitative methods based on surface remission of light are utilized. The profile of a regular and an irregular line shape demonstrates such quantitative methods (see Figure 4). Visibility with the naked eye is limited to that portion of the protein that is bound to the top of the membrane, as well as a thin adjacent layer of the membrane. To quantitate the total protein bound within the entire membrane matrix, a densitometric analysis of transmission light is suggested.
Correlation with Membrane Properties. The challenge for membrane manufacturers today is to understand precisely why some lots of cellulose nitrate membranes exhibit sharp protein lines and why some others will give more diffuse and fuzzy lines (see Figure 5). Apart from variations due to reagents, identification of variations that can influence the protein line shape, and the possible solutions to these variations, is an important subject.16,17
Surface Quality
It is already known that with an unbacked membrane, the side of the membrane that has been in contact with the casting belt (see Figure 2) is much smoother and cleaner than the evaporation or air side.
This belt side of the membrane indeed always gives sharper protein lines. However, due to the very weak tensile strength of unbacked membranes, most IVD manufacturers prefer to work with supported membranes that are cast directly onto polyester film. The plastic film is inserted onto the belt at the head of the machine before the slit coating equipment applies the polymer mix. With these supported membranes, only the air side is available for line application. The membrane manufacturer’s task is to provide a surface as homogeneous and clean as possible.
A common phenomenon during the evaporation casting phase is the deposition of dust or powder. This dust consists of small particles of cellulose nitrate that is precipitated independently from the main membrane layer. Dust may be located directly at the surface or slightly incorporated inside the membrane layer. Protein line formation is always affected, since these particles are not homogeneous in size and distribution, and may even migrate with the applied liquid sample. Manufacturers today are applying two practices to overcome this problem.
- Avoid the formation of dust during the membrane manufacturing process.
- Remove the dust at the end of the membrane manufacturing process.
Since the formation of dust depends on the raw material as well as on slight variations of the process conditions, the second approach is probably more effective.
Surfactants
The use of surfactants is indispensable since cellulose nitrate is a hydrophobic polymer. All surfactants interact with proteins as well as with the membrane, and thus decrease the binding characteristics. Membrane and IVD manufacturers are very secretive about the surfactants that they use. The only practical strategy is to develop the right cocktail, which is generally a mix of different types of surfactants and additives that will increase overall protein binding and maintain optimal wicking speed. For the same reason, another approach would be a surface modification of the membrane, either by adding cross-linked polymers or by physical treatment.
Conclusion
The line test is an extremely useful tool to qualify membranes for diagnostic applications. Optimum reproducibility of membrane quality should be the first priority. Since the demands of the IVD industry will continue to grow, membranes with higher binding capacity, even for small proteins, and faster migration speeds will be required. Although developing a membrane with both higher binding capacity and faster migration speeds may seem like competing goals, it is precisely this challenge that membrane manufacturers must address in order to do their part to grow the IVD rapid test industry.
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15. TM Klewitz, "Proteinbindung an Diagnostikmembranen, Thesis for diploma examination, University of Hannover, Germany, (2000), 9.
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Hans H. Beer is senior scientist in the R&D department of membrane evaporation process, Eric Jallerat is international sales and marketing manager for diagnostic membranes, and Karl Pflanz is head of the R&D departments of membrane evaporation at Sartorius Corp. (Goettingen, Germany). The authors can be reached via hans.beer@sartorius.com, eric.jallerat@sartorius.com, and karl.pflanz@sartorius.com, respectively. Timo M. Klewitz is a PhD student at the University of Hannover, Germany, and can be reached via timo.klewitz@sartorius.com.
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