Troubleshooting protein binding in nitrocellulose membranes
Part 1: Principles
Kevin D. Jones
Developers of membrane-based assays should have a firm grasp
on the various factors that can influence protein
binding—including those inherent in the materials and processing
used for their tests.
The number of membrane-based rapid immunochromatographic devices on the market is
continuing to increase at a very quick pace. Major factors that are contributing to this growth
include improvements in conjugate technology and a growing understanding among product
developers of the general design principles involved.
Although today's immunochromatographic devices come in a wide variety of designs with a diverse
assortment of housings, most commercially available tests are based on one of two simple formats.
The most common format is the lateral-flow or dipstick design, which has become familiar through
its use in physician-office assays as well as in over-the-counter tests (e.g., Unipath's Clear Blue
pregnancy test). A less widespread format is the flow-through or transverse-flow design, which
requires greater operator skill and is therefore usually restricted to professional use (e.g.,
Medmira's rapid HIV screen).
Figure 1. Achieving a crisp, clear test result, such as in the
samples shown here, depends on correct binding of the capture
reagent to the membrane.
Regardless of the format being used, achieving a sensitive and reproducible test requires the
manufacturer to have an efficient procedure for applying the capture-line reagent. Companies
involved in the rapid diagnostic industry have been active in publishing information about how to
optimize capture-line application.1–5 This article offers further aid to product developers, discussing
the basic principles involved in applying protein capture lines to nitrocellulose membranes, and
highlighting some of the common problems that can be encountered during the development of an
immunochromatographic assay. Because the problems associated with protein binding are more
prevalent in lateral-flow assays, this article will focus especially on issues relating to such systems.
The Importance of Protein Binding
In immunochromatographic assays, the primary function of a protein applied to a membrane is to
act as a capture reagent for the target analyte in a sample. Because the test result is totally
dependent upon achieving a good binding of the capture reagent to the membrane, the importance
of achieving a high and consistent level of protein binding cannot be overstressed (see Figure 1).
Despite the considerable amount of research that has been conducted since nitrocellulose was first
used as a protein-binding membrane, the exact mechanism of that binding remains unknown. 6 It is
known that a number of forces are at work—specifically, hydrophobic interactions, hydrogen
bonding, and electrostatic interactions—but a clear understanding of the exact effect and
significance of each force has remained elusive. Two reasonable models have been proposed. The
first model suggests that proteins are initially attracted to a membrane surface by electrostatic
interaction, while long-term attachment is accomplished by a combination of hydrogen bonding
and hydrophobic interactions. Although extremely difficult to prove, this model of the interaction
fits the published experimental data and is often the accepted mode of
interaction.1, 7–11
Figure 2. Problems with protein binding are typically visible in the
capture line of an assay's test result, as in these examples.
A second model suggests that the initial attachment of the protein is caused by hydrophobic
interactions, with long-term binding accomplished by electrostatic forces. This model also agrees
with much of the published data. However, the electrostatic partition mechanism may not provide
a full explanation for the long-term stability conferred on protein attachment by drying or the use
of an alcohol fixation step.3,6
Whatever the balance of forces responsible for protein binding, it is widely agreed that product
developers should consider all such forces when they are seeking to optimize the binding of
proteins to a particular membrane. Such considerations will inevitably have implications for both
the selection of materials to be used, and the ways that they will be processed. For
instance, if the product developer selects a buffer that too greatly reduces either
hydrophobic or electrostatic interactions, the level of protein binding could be
dramatically reduced. Similarly, it is widely recognized that adequate drying of the
membrane after protein application is an important practice for ensuring the
long-term stability of the protein–membrane bond.1–4, 6
Figure 3. A weak capture line indicates that the amount of protein bound to
the membrane is too low.
The manufacturer's selection of materials can have an effect on the binding of proteins to
nitrocellulose membranes. Materials that interfere with protein binding can be divided into three
general types: nonspecific proteins, materials that interfere with electrostatic interactions, and
materials that interfere with hydrophobic interactions. Commonly used materials that reduce
protein attachment include those that compete for binding sites, such as the classic bulking
proteins (e.g., BSA, animal sera), as well as those that interfere with hydrogen bonding (e.g.,
formamide, urea) and those that interfere with hydrophobic bonding (e.g., Tween, Triton, or Brij).
Man-made polymers such as polyvinyl alcohol (PVA), polyethylene glycol (PEG), and polyvinyl
pyrrolidone (PVP) can also interfere with protein binding. Their mode of action may be a
combination of effects that inhibit one or more of the forces essential to protein–membrane
binding.
If an insufficient amount of protein binds to the membrane, or if the protein does not bond to the
membrane with the necessary strength, some significant problems can arise. These problems are
typically visible in the capture line of an assay's test result (see Figure 2). If the amount of protein
bound to the membrane is too low, the resulting capture line will be weak and test sensitivity will
be reduced (see Figure 3). If binding is inefficient, the protein can diffuse before
finally becoming immobilized on the membrane. The resulting capture line will be
broad and weak instead of crisp and clear, making test results difficult to
interpret. In extreme cases where the physical attachment of the protein to the
membrane is too weak, the passage of analyte proteins and surfactant solutions
can actually wash capture reagents off the membrane. In such cases the assay
will display a broad line—or no clear line at all—again making it difficult to
interpret the test results (see Figure 4).
Figure 4. A diffuse capture line can result when the capture reagent is
washed away by the passage of analyte proteins and surfactant solutions.
Problems such as these are regularly seen by product developers in the IVD industry, and can
significantly slow the development of a successful immunochromatographic assay. To understand
how to go about resolving such problems, developers should first have a firm grasp on the various
factors that can influence protein–membrane binding, including those inherent in the materials and
processing used for their tests. These elements will be discussed in the first installment of this
article. Typical techniques for solving such problems will be given in the second installment, which
will appear in a future issue of IVD Technology.
Factors That Influence Protein Binding
When investigating the binding of protein capture reagents to nitrocellulose membranes, product
developers should consider each of the following five critical areas that can have an effect on the
binding mechanism.

The application buffer in which the capture reagent is dissolved.

The membrane to which the capture reagent is applied.

The capture reagent itself.

The system used for applying the protein to the membrane.

The ambient humidity at the time of protein application.
Although many development labs do a good job of studying and characterizing the application
buffers and membranes used in their tests, they are less likely to fully investigate or optimize the
capture reagents and application systems they employ. Such an omission is often due to the fact
that the latter elements are frequently considered set even before the beginning of the
development process, leaving little opportunity for changes to be made. With those factors out of
consideration, product developers often have no choice but to focus on optimizing the other
elements that are still within their discretion.
Capture Reagents. The proteins used as capture reagents vary from test to test. However subtle
their differences, no single capture reagent is absolutely identical to another. Perhaps more
important, different proteins exhibit varying levels of attachment to different membranes (see
Figure 5).5 The process of optimizing binding is most straightforward with a monoclonal antibody,
where the protein is a homogeneous material. Optimization is more difficult in the case of
polyclonal antibodies because there are a variety of epitopes present, and ideally each requires
slightly different binding conditions. Species such as IgA or IgM can present an even greater
challenge because of the potential for structural or steric problems. Other proteins such as BSA,
protein A, or protein G can cause significant difficulties due either to their chemistry or their size
(large molecules are more likely to remain attached to a solid phase than smaller ones).
Application Equipment. Although systems used to apply capture reagents can also present
problems, most commercially available equipment has both advantages and disadvantages.
Variables can include the ability or inability to dispense measured volumes; capacity to handle
strips, sheets, or membranes; speed of application; and postapplication handling of strips. The
best solution is for the manufacturer to find an application system that satisfies the most
significant practical issues, such as raw material limitations and system capacity. Other factors can
then be optimized for that particular application system.
Figure 5. Comparative binding of IgG and albumin to a range of nitrocellulose
membranes from different manufacturers. To replicate actual test conditions, data were
generated using a flow-through system where the sample was applied to the membrane
surface and pulled through the membrane by vacuum.16 Although the more common test
method is to incubate the membrane with the protein solution, that method permits
protein molecules to stack up within the pores of the membrane, resulting in the
formation of a protein multilayer instead of a protein monolayer.1 The traditional test
method thus results in artificially high levels of protein binding that are
unrepresentative of actual use in rapid immunochromatographic tests.
Ambient Humidity. The humidity at the time that the capture line is applied can have a
significant effect on the quality of the line, especially when spray systems are used. If atmospheric
humidity is low a static charge can collect on the membrane, which can result in satellite spots
when the protein is sprayed onto the membrane surface. Low humidity can also cause the
development of hydrophobic patches on the membrane surface. By contrast, extremely high
humidity can result in very rapid wicking of the applied protein, causing wide or diffuse capture
lines. In general, the optimal humidity in which to apply proteins is between 45 and 65% RH. To
ensure even properties throughout the feedstock, the membrane should be allowed to equilibrate
with the atmosphere before application. The optimal equilibration time should be determined by
experimental investigation.
Optimizing the Application Buffer
Because protein capture reagents vary, maximizing the binding of a given protein may also require
buffer conditions that differ from those appropriate to another protein. There are two important
factors that need to be optimized through modifications to the application buffer.

The solubility of the protein (i.e., the amount of protein physically available for attachment).

The stability of the protein molecules (i.e., whether they tend to agglomerate or to stay in
solution).
To ensure that sufficient protein is available in the applied capture line, it is first essential that the
capture protein be soluble in the application buffer. In order to confer enough solubility to enable
the protein to be dissolved, it is necessary to have some ions present in the application buffer.
Although the ionic strength of the buffer can help to control the pH of the capture reagent, it also
interferes with electrostatic interactions essential to protein binding. It is therefore important to
determine the lowest possible ion level for the buffer that will result in a sufficient concentration of
capture protein in solution.
If the molecules of a given protein concentration are stable in solution, they will tend to remain so.
But if it is energetically favorable for the protein to partition onto the solid phase, then a greater
proportion of protein will attach than if the protein is stable in solution. Such an energy state can
be induced through the use of destabilizing or coprecipitating agents. However, too much
correction in this direction can cause other problems. If the protein precipitates before it can be
applied to the membrane, for instance, the entire system will become highly unstable and almost
totally irreproducible. The amount of dissolved protein remaining for attachment to the membrane
will thus be dramatically reduced. Precipitates may also cause problems by blocking the application
equipment or clogging the pores of the membrane. There are some cases in which obtaining a
reasonable level of binding may make it necessary to cause the protein to precipitate during
application, but these are exceptions to the general rule.
As the above analysis suggests, protein binding can be altered by adjusting the properties of the
application buffer (see Figure 6). Key properties that can be usefully modified include the buffer's
ionic strength and acidity, and the level of coprecipitating agents employed.
Ionic Strength. Within a defined range of ionic strength, the solubility of a typical protein
increases in direct proportion to the salt content of the application buffer. Because it is desirable to
minimize the molecular stability of a capture protein in solution, the ionic strength of the solution
should be kept as low as possible. Doing so will increase the speed of protein binding. Developers
should also be aware that high salt concentrations can cause precipitation of proteins, and that the
presence of large quantities of salt during drying can interfere with the sensitivity and stability of
the test.
Acidity. The pH level of an application buffer can have a significant effect on its properties. The
solubility of a typical protein is at its minimum at its isoelectric point. Since developers are aiming
to minimize the molecular stability of the capture protein in solution, the ideal pH of the application
buffer should therefore be at about the isoelectric point of the capture protein being used.
Coprecipitating Agents. When modifying an application buffer, developers may choose to add a
destabilizing or coprecipitating agent in order to reduce the stability of the protein molecules in
solution. The action of such coprecipitating agents relies on the differing stability that the fc and
f(ab) regions of the IgG molecule have toward the agents used.11 The structure of the fc region is
far more likely to be degraded by the action of coprecipitating agents. Partial destabilization of the
fc regions leads to the exposure of more-hydrophobic groups that are normally hidden within the
protein structure. Thus, regardless of which mechanism is accepted for the binding of proteins to
nitrocellulose, the increase in protein hydrophobicity resulting from the use of such coprecipitating
agents will improve protein binding.
Figure 6. Varied results from capture lines of 1mg/ml mouse IgG applied using different
buffers: (a) 10 mmol phosphate, pH 7.2; (b) 10 mmol phosphate + 3% methanol, pH 7.2;
(c) 10 mmol phosphate + 150 mmol NaCl + 3% methanol, pH 7.2; (d) 50 mmol
phosphate + 150 mmol NaCl + 1% BSA, pH 7.2; (e) 50 mmol phosphate + 150 mmol
NaCl, pH 7.2; (f) 50 mmol phosphate + 150 mmol NaCl, pH 6.0. All samples were
detected by a 40 nmol gold-conjugated goat antimouse IgG antibody.
The most commonly used coprecipitating agent is alcohol, which can be recommended for a
number of reasons. The presence of alcohol helps to rewet the membrane, reduces any static
charge it may have, and has a destabilizing effect on the protein in solution. Levels of between 3
and 5% methanol can give considerable improvement in the performance of a membrane used for
an immunoassay.3
The use of alcohol to improve protein binding to a solid phase has been known for several years in
the production of ELISA plates, and is now regarded as a standard protocol. 11,12 The influence of
aliphatic alcohols on binding in nitrocellulose membranes was first reported in 1980, while a 1%
isopropanol solution is widely used as a fixing solution in protein blotting experiments. 6,13
Although other materials such as diethylaminoethyl or ammonium sulfate can sometimes have
beneficial effects when used as coprecipitating agents, they are generally less desirable than
alcohol. Even small variations in the concentration of these types of materials can have severe
effects on the degree of protein precipitation. For this reason, precipitating agents other than
alcohols should generally not be used.
Considering the points outlined above, a buffer comprised of 10 mmol phosphate +3% methanol
pH 7 is suggested for initial development studies. Although such a buffer will not prove optimal for
all applications, it offers a very good starting point for the development process.
Membrane Effects
The membrane itself has a significant effect on the protein binding observed in a rapid assay, with
three key factors affecting membrane performance:

Pore size.

Posttreatments.

Membrane type.
Because of the wide range of potential capture reagents, no single membrane will work optimally
for every assay. The level of protein binding can vary dramatically among different types of
membranes (see Figure 5). Unfortunately, this means that product developers must reinvestigate
and optimize their membrane selection for each assay they develop. However, the potential
improvement in test performance and assay reproducibility is sufficient compensation for the
additional work involved.
Pore Size. Developers of lateral-flow immunoassays should treat
supplier references to pore size with caution. The actual pore size
of a membrane depends on the method used to measure it, and
since different manufacturers use different measuring techniques,
any two membranes with the same nominal pore size could differ
significantly if measured by a constant technique (see Figure 7).
Figure 7. Pore size data for nitrocellulose membranes
based on data from a Coulter porometer.
Pore sizes are usually measured in the filtration direction, that is, through thickness of the
membrane. But the size and shape of pores in the filtration direction may have no relation to the
size and shape of pores in the lateral direction (that is, along the length of the membrane). For a
lateral-flow assay therefore, the conventional method of quoting pore size is not really relevant.
Moreover, if a plastic cast membrane is used, measuring pore size in the filtration direction is
physically impossible because of the presence of the film backing. In such cases, the pore sizes
quoted by suppliers are often no better than best estimates based on lateral wicking data.
Product developers can use nominal pore size—cautiously—to differentiate membranes from a
single manufacturer. But it is not recommended that such information be used to specify pore size
for membranes from another manufacturer. Nominal pore size generally has no standard meaning
in terms of protein binding, particularly for lateral-flow assays, and developers are better advised
to screen a range of membranes when they begin the development of a new assay.
Although nominal pore size has little real importance, the lateral pore size and structure of a
membrane does have a significant effect on its suitability for use in lateral-flow assays. Within any
range of nitrocellulose membranes, as pore size decreases the protein binding capacity of the
membrane increases because of the related increase in available membrane surface area. 1 The
approximate surface area for membranes of different pore sizes can be estimated by looking at the
surface area ratio (SAR) for each material.3 The SAR represents the ratio of actual available
surface in the pores of the membrane to the area of membrane used (see Table I). 14 Another
phenomenon of importance is that as a membrane's pore size decreases, the lateral wicking rate
of the membrane also decreases (see Table II). 15 A slower wicking rate increases the effective
sensitivity of a test because it permits reagents to spend a longer time in the capture zone.
Figure 8. Water present during the application of posttreatments
can make sections of the membrane hydrophobic, resulting in
striations or intensity variations in the capture line.
The combined effect of these two phenomena is that greater relative sensitivity is achievable by
using membranes with a smaller pore size. Thus, as a general guideline, a developer who is most
concerned with the ultimate sensitivity of an assay should select a membrane with the smallest
possible pore size; while a developer who is primarily concerned with the wicking speed of an
assay should select a membrane with a larger pore size. Whatever their needs, developers can
best find the optimal membrane for their tests by evaluating a variety of possibilities during the
early stages of product development.
Posttreatments. Following manufacture, nitrocellulose membranes routinely receive
posttreatment to remove dust (unincorporated polymer left on the surface of the membrane after
manufacture) or to modify their rewetting characteristics. In either case, there is the possibility
that such posttreatment may introduce trace chemicals or other substances that are not
nitrocellulose, and that may have an effect on the performance of the finished test device.
In general terms, the manufacturer should always know what additional substances may be
present in the membrane, in what concentration they are present, and how to measure their levels.
Depending on what additional materials are present, significant effects may be observed in the
level of protein binding, the flow rate of the membrane, and in the effects of aging on the
membrane.
Industry at large generally accepts the practice of posttreating nitrocellulose membranes in order
to preserve their wetting properties. However, there is disagreement about whether membranes
should be treated with a wetting agent before they are delivered to the customer or later in the
manufacturing process, after the capture line has been applied to the membrane. Purchasing
membranes that have already been treated can be an attractive alternative, because such
materials can reduce the amount of processing that the test manufacturer must perform. Such
posttreated membranes can be used directly off the shelf, thereby eliminating the costs of
additional equipment to perform a treatment step after protein application, and the time required
to do so. Before deciding to accept such treated membranes, however, developers should consider
the following issues that can make them less suitable for some applications.
One major disadvantage of posttreating a membrane before capture line application is that the
wetting agent can leach off or migrate through the membrane, with results that can become
especially noticeable when the membrane is stored for an extended period before use. Changes in
the concentration of the wetting agent can affect the protein binding properties of the membrane,
as well as its wetting properties and lateral wicking rate. The shelf life of a test can thus become
dependent on the concentration of wetting agent applied to the membrane, possibly some
significant time before the test is actually manufactured, and perhaps unknown when the
membrane is used for test production. On the other hand, when the manufacturer performs such
posttreatment in-house after purchase, the assay developer can record the level of rewetting
agent in the membrane and can conduct adequate aging studies on the material. This enables the
developer to create appropriate posttreatment protocols that optimize the long-term storage and
use of the membrane.
In untreated nitrocellulose membranes, the hydrophilicity of the material is a direct function of its
pore structure. But when such membranes are posttreated with a hydrophilic agent, it is the
posttreatment that governs the hydrophilicity of the material. If the posttreatment migrates during
storage or is washed off by the sample, the comparative performance of membranes with the
same nominal pore specifications can thus change quite dramatically. Initial quality control (QC)
testing can enable the manufacturer to determine the combined effect of the membrane's pore
structure and hydrophilic posttreatment, but as the posttreatment ages or is removed the
performance of the membrane will become increasingly dependent upon its pore structure, and the
results of the initial QC tests will become invalid. Product developers can ensure a less-variable
product by using membranes whose pore structure is consistent between manufacturing batches,
and which have not undergone posttreatment with hydrophilic agents.
Since most posttreatment agents are water soluble, any water present when the capture line is
applied can wash the posttreatment away from the point of capture line application. This can result
in a portion of the membrane being without wetting agent, and therefore highly hydrophobic,
making the capture line inaccessible to the sample and conjugate. These effects can dramatically
affect the readability of the assay. Frequently, such hydrophobicity causes the sample to pass
unevenly through the capture line, resulting in striations or intensity variations (see Figure 8). In
extreme situations the capture line can appear white against a colored background. Applying
posttreatments after capture line application can avoid these pitfalls.
Assay developers should weigh the time and cost benefits of using an already posttreated material
against the consistency and long-term stability advantages of a material that contains no
surfactant posttreatment.
Membrane Type. Independent of the pore size of a membrane or any posttreatments applied to it,
the type of membrane used also has a significant effect upon the protein binding levels observed in
a test. Figure 5 shows the comparative levels of immunoglobulin and albumin binding for a range
of nitrocellulose membranes from different manufacturers and with varying nominal pore sizes. 16
Both series of data display variations in binding levels that have little to do with the nominal pore
sizes of the membranes. Comparison of the figures also demonstrates that the membranes with
the best immunoglobulin binding do not always give the best albumin binding. 2,16 Relative protein
binding levels can therefore be influenced both by membrane formulation and the source of
manufacture.
For a series of membranes with a fixed surface area, the level of protein binding is a function of
polymer type and the presence of any treatment agents that affect the surface energy of the
membrane. The base polymer for membrane production is available from a number of commercial
sources; but each source's material has slightly different properties, and a variety of different
membrane treatments are used by membrane manufacturers. It is always advisable for product
developers to conduct experiments to evaluate the relative protein binding performance of any
membranes they are considering for their tests.
Nominal Pore Size (µm) Surface Area Ratio (SAR)
3
110
5
98
8
66
12
63
Table I. Surface area ratio data for Whatman
nitrocellulose membranes.14 Data produced by
BET surface area measurements using nitrogen.
Membrane Aging. Unless they have been treated with a rewetting agent, freshly manufactured
nitrocellulose membranes will retain moisture on their surfaces. This moisture has been measured
as being between 5 and 10% by weight.1,17 Traditional theories concerning the aging of
nitrocellulose membranes have largely assumed that this retained moisture is volatile; thus as the
membrane ages it loses moisture and becomes hydrophobic, statically charged, and brittle.1,15 The
actual situation may not be so simple. Some research on nitrocellulose aging seems to dispute the
conclusion that the moisture retained by nitrocellulose membranes is volatile, and recent studies
have shown that effects similar to hydrophobicity can be achieved during storage even at very
high humidity levels.13,17
If a membrane has been treated with a rewetting agent, then the storage properties required to
maintain the stability of that membrane will depend on the characteristics of that particular
material. When storing nitrocellulose membranes, it is important that they be protected from
exposure to organic solvents (which can cause hydrophobicity) and strong sunlight (which can
result in chain scission). At present, the best conclusion that can be drawn is that nitrocellulose
membranes, if stored under the correct conditions (45 to 55% RH), will remain stable for at least
two years. Membranes that have been blocked after the application of the capture reagent will
remain stable for longer periods.
Nominal Pore Size (µm) Time to Rise 4.5 cm (sec)
3
245 ±35
5
185 ±25
8
140 ±20
12
100 ±20
Table II. Typical wicking rate data for Whatman
membranes.15 The tests were performed using
water as the test liquid at 25°C and 49% RH.
Optimizing Membrane Flow
The flow rate of a membrane used in an immunoassay is one of the key influences on test results.
For a given combination of membrane, capture reagent, and analyte concentration, the sensitivity
of the test will increase with decreasing flow rate. In effect, the apparent concentration of the
analyte increases as the flow rate decreases. The relationship between the lateral wicking rate of a
membrane and the apparent analyte concentration follows an inverse square law:
apparent analyte concentration
1/(lateral wicking rate)2
Thus, a doubling in the lateral wicking rate will reduce the apparent analyte concentration to 25%
of the previous value.
Blocking Reagents. Nitrocellulose membranes are naturally hydrophobic. The wicking of water
into a nitrocellulose membrane is due to the presence of interstatial moisture. 1 The hydrophobicity
of the membrane can give rise to three major problems:

Nonspecific interaction of a hydrophobic analyte with the membrane can generate
significant nonspecific signal.

Loss of interstatial moisture can cause long-term storage difficulties.

The rate of rewetting and lateral flow can be too low for effective use in rapid assays.
Material
Typical Working Range (%)
Tween 20
0.01–2
Triton X-100
0.01–2
PVA (15 kDa)
0.1–5
PVP (33 kDa)
0.1–7
PEG (20 kDa)
0.05–3
Brij
0.05–3
Table III. Materials that show efficient
membrane blocking properties.
The purpose of blocking a nitrocellulose membrane after application of the capture reagent is to
reduce or eliminate these problems. Ideally, the blocking materials that a developer selects should
help to ensure the production of a stable and reproducible test system. They should simultaneously
reduce the level of nonspecific background staining, remain bound to the membrane during
prolonged storage, and achieve even and consistent rewetting upon sample application at a rate
appropriate to the assay being developed.18 They should also be free from interference with the
interaction of antibodies and antigens at the capture line, where such interference can
substantially reduce the signal intensity of the test.
Figure 9. The variation in nonspecific binding observed with a range of mixed polymer
and surfactant blocking agents.
Considering these key features of the ideal blocking system, the choice of a blocking agent often
requires developers to seek a compromise among four performance factors:

Lateral wicking rate.

C
a
p
t
u
r
e
l
i
n
e
intensity.

Capture line width.

Nonspecific background intensity.
Unfortunately, such performance factors are often mutually exclusive. For example, blocking
agents that reduce nonspecific interactions usually also reduce the amount of specific signal, and
agents that increase the wicking rate of the test usually also reduce test sensitivity and potentially
increase the width of the capture line. In general terms, there are three classes of blocking agents:

Surfactants.

Man-made polymers.

Proteins.
The best results can generally be obtained by using a combination of materials from at least two
separate classes, normally by mixing surfactants and man-made polymers (see Table III). Proteins
are usually not the most efficient materials; although they can help to reduce the levels of
nonspecific background signal, they also dramatically reduce the lateral wicking rate of the test
system (see Figures 9 and 10).19
Blocking Methods. When the supplier of a nitrocellulose membrane has posttreated it with a
rewetting agent, it is generally unnecessary for the test manufacturer to repeat this step. When
blocking is required, it is generally performed in one of three ways. The first method is to immerse
the membrane in a bath of the blocking solution, allowing it to saturate the membrane with the
desired concentration of the blocking agent. The second method is to dry a high concentration of
the blocking agent onto a lower part of the test device (typically the conjugate pad or the sample
application pad). When the sample is applied it redissolves the dried blocking agent, which then
flows with the sample and blocks the membrane in situ. The third method is to dilute the sample in
a running buffer that contains the blocking agent. This method offers good results, but requires the
user to perform an extra step that is undesirable in many over-the-counter tests.
Figure 10. The variation in nonspecific background observed with a range of single
material blocking agents.
Whatever type of blocking system is chosen, the developer should perform adequate shelf-life
studies to assess the stability of the test. Although accelerated aging studies are a useful means of
evaluating the potential shelf life of a test, care must be taken when interpreting the results of
such tests. A typical conversion factor used for accelerated aging studies is to multiply the
observed life at 37°C by a factor of 10. Thus four weeks at 37°C would be the equivalent of about
40 weeks at room temperature.17 Although industry generally considers such a conversion factor to
be a reliable rule of thumb, it is not a guarantee of stability.
Conclusion
This installment has looked at the major factors that can influence the binding of proteins to
nitrocellulose membranes. The second installment of this article, which will appear in a future issue
of IVD Technology, will provide more-specific examples of how to overcome protein binding
problems that are commonly encountered during the development and manufacture of an
immunochromatographic test system using nitrocellulose membranes.
Continue to Part II of this article.
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Schleicher & Schuell, 1991).
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Schuell, 1997).
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1996).
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1997–1998).
5. The Latex Course, 1994 (Fishers, IN: Bangs Laboratories, 1994).
6. E Harlow and D Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor, NY: CSHL Press,
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1981).
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14. R Bowen, private communication with author, Swansea, UK, February 3, 1998.
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Comparison of Commercially Available Membranes for a Novel Flow-Through Immunoassay,"
poster no. 21 (presented at the 1998 Annual Meeting of the American Association for Clinical
Chemistry, Chicago, August 2–6, 1998).
17. Unpublished results (Maidstone, UK: Whatman International, 1998).
18. AM Campbell, Monoclonal Antibody and Immunosensor Technology (Amsterdam, The
Netherlands: Elsevier, 1992).
19. KD Jones and AK Hopkins, "Evaluation of the Efficiency of a Range of Membrane Blocking
Agents for Nitrocellulose Membrane Based In Vitro Diagnostic Devices," poster no. 3 (presented at
the 1998 Annual Meeting of the American Association for Clinical Chemistry, Chicago, August 2–6,
1998).
Kevin D. Jones, PhD, is manager, diagnostic technology, at Whatman International Ltd.
(Maidstone, Kent, UK).
Continue to part II of this article.