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.

    市面上的基于膜快速免疫层析装置的数量以非常快的速度增长。这个增长的主要原因包括金标技术的提高和产品开发者之间了解的增多。

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.

    虽然今天的免疫层析装置在不同的分类中有很多种产品设计，上市最多的试剂是基于两种简单形式之一。最普通的形式是侧向流动或试纸条设计，通过在医生诊所检测和直接销售给用户的方式，使得它很常见。另一种较少见的形式是渗透设计，需要较强的操作技巧，因此只限于专业使用。

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.

    不管用到哪种形式，要达到一个灵敏的特异的试剂需要生产者有有效的程序来使用捕获线试剂。快速诊断行业中的企业经常活跃在关于怎样优化 捕获线的出版物上。这些文章产品开发商提供更进一步的帮助，讨论蛋白捕获线试剂固定到硝酸纤维素膜上的基本原理，突出免疫层析试剂开发遇到的共同问题中的一些。因为在侧向流动试剂中，关于蛋白固定的问题很普遍，所以这篇文章主要集中这些系统相关的问题。

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).

    在免疫层析试剂中，应用在膜上的蛋白的首要作用就是作为样品中目标分析物的检测试剂。由于检测结果完全地依靠检测试剂在膜上达到良好的固定效果，所以完成蛋白固定的高且一致水平的重要性不言而喻。

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

    自从NC膜首次被用于蛋白固定以来，尽管以NC膜为基础的研究大量的进行过，但是结合的确定机理仍然不知道。比较为人所知的作用力有疏水相互作用力、H键、静电相互作用力等，但是对确定作用力的理解和各作用力的贡献还不是很清楚。目前主要有两种假说：第一种认为蛋白质与膜的开始结合由静电相互作用产生，然后依靠他们之间的H键和疏水相互作用来维持它们间长时间的结合。虽然很难证明这种假说，但是这个模型拟合发表的试验数据，通常被模型所接受。

    以上两种假说尽管都有实验数据支持，但是实验也表明，任何能够影响疏水相互作用、H键、静电相互作用的因素，都将影响到蛋白质与膜的结合。

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

    第二种认为蛋白质与膜的开始结合由疏水相互作用产生，而它们间长时间的结合由静电相互作用来完成。这种模型也能和很多发表的数据一致。然而静电机制对用干燥或乙醇固定步骤来固定蛋白的现象不能给出很好的解释。

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

    不管蛋白固定的结合力是如何平衡的，产品开发者在寻求蛋白固定的优化条件时，应该要考虑所有这些作用力。这些考虑包括使用材料的选择和加工它们的方法。例如，产品开发者选择了一种明显降低疏水作用和静电作用 的缓冲液，蛋白固定的水平可能会急剧下降。同样，普遍认为蛋白应用后对膜足够的干燥，对确保蛋白－膜之间长期稳定性是重要的步骤。

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).

    如果膜上蛋白质的量不足或者膜与蛋白质不能很好地结合，将会出现一些重要的问题。从捕获线的试验结果上我们可以清楚地看到这些现象（如图2）。如果膜上的蛋白质量偏低，捕获线将偏弱并且灵敏度也会降低（如图3）。如果蛋白质失效，蛋白质在膜上固定以前将会发生扩散，捕获线将变得宽而弱，这会结果变得难以说明问题。举一个极端的例子，如果说膜上蛋白质的物理吸附能力足够弱，那么分析蛋白质和表面活性溶液通过膜时将会冲走其上面的反应物，实验结果将产生一条很弱的线或者说根本就是一条模糊的线，这样的试验结果很难说明问题（见图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.

    在研究蛋白检测试剂固定在NC膜上时，产品开发者应当承认以下五个方面，可能会影响条带的机制：

• 溶解检测试剂的缓冲液。

• 检测试剂作用的膜。

• 检测试剂本身。

• 用于蛋白质与膜结合的系统。

• 蛋白使用时的环境湿度。

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.

    虽然很多开发实验室在学习和描述用于它们试剂的缓冲液和膜方面做的很好，但是他们还是不大可能全部研究或优化他们用到的检测试剂和应用系统。这些忽略通常是因为后者在开发之前就经常被考虑到，在开发中很少有机会被改变。由于那些忽略的因素，产品开发者往往没有机会并集中优化在他们考虑范围 内的其他因素。

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).

捕获试剂
    用来作为捕获线的蛋白因为不同的测试目的而必须相应的变化。无论他们之间差异如何微妙，没有一种蛋白质会与另一种蛋白质完全一样的。更重要的是，不同的蛋白质在不同的膜上的结合水平有很大的差异。因此，在优化过程中，最好是直接用单克隆抗体（如果测抗原），这里蛋白是均一性物质。而用多克隆抗体主要问题是它们有不同的抗原决定基，各种抗原决定基的抗体与膜的最佳结合条件都有细微的差别，这样就增加了优化的难度。IgA、IgM这些分子由于结构或位阻问题，优化的过程会面临更大的挑战。而象BSA、Protein A、Protein G等大分子蛋白质由于它们的化学性质及分子量的缘故，调节它们与膜的结合特性就更加困难（分子量越大，蛋白质越易吸附到固相材料上）。

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.

环境湿度

    环境湿度条件对点膜过程非常重要，它会严重影响C、T线的质量，特别在喷膜系统在使用过程中。如果空气湿度太低，静电荷容易聚集在膜上，这时点膜就容易产生斑点；而这时测试，膜上就容易产生疏水斑。相反，空气湿度太高，膜上的毛细作用加强，这时点膜就容易引起C、T线变宽甚至扩散。一般来说，最佳的环境湿度应保持在45~65%RH。而且，为了保证所有要用的膜材料有均一的特性，点膜前应把膜放到工作环境平衡一段时间。最佳的平衡时间应由实验来确定。

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.
    因为作为捕获线的检测试剂都不一样，优化给定蛋白固定的缓冲液是不同于其他蛋白的。通过改变所用的缓冲液，需要优化的两个重要因素是：

• 蛋白质的溶解性

• 蛋白质分子在溶液中的稳定性

    确保足够的蛋白被用到检测线上，首先检测蛋白要溶于应用缓冲液中。因为要保证有效的蛋白质溶解度，溶液里就要有一定的离子强度；但是离子强度高了，正负离子又会干扰蛋白质与膜的相互结合。因此，最重要的一点是如何决定一个尽可能低的离子强度溶液而又能保证足够有效的蛋白质溶解在缓冲液里。

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.

    如果蛋白质在给定的浓度下物理特性稳定，则它将趋向于溶解在溶液里。但是若它的能量状态有利于形成固相，那么吸附到膜上的蛋白比稳定溶解在溶液里的多。这种能量状态可以通过加入去稳定剂和沉淀剂来形成，然而，如果这类试剂的作用过量了，也会产生一些其它的问题。例如，若蛋白在用到膜上之前就发生沉淀，那么整个试剂系统就高度不稳定且几乎完全不可再生，可用来吸附到膜上的溶解蛋白的量就因此急剧下降。且溶液中的沉淀也可引起如堵塞使用中的设备及闭塞膜上的微孔等现象。有些情况下为了获得一合适吸附水平的蛋白浓度，在生产中或许有必要使蛋白质沉淀。这些对一般规律来说是例外的。

    从以上的分析表明，蛋白质与膜的结合能力可以通过调整它所处的缓冲系统的特性改变，关键的是缓冲液的离子强度、pH值及所用沉淀剂的水平。

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.

离子强度 

    在一定范围内，一般蛋白质的溶解性将随着溶液离子强度升高而增大，为了使溶液中的蛋白质分子的稳定性降至最低，因此，溶液中应保持尽可能低的离子强度。这样的话使蛋白质与膜的吸附固定的速度上升。开发者也应注意到高盐浓度也会引起蛋白质的沉淀，而且在点样后膜在干燥时，大量盐的存在将干扰测试的灵敏度和稳定性。

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.

酸度 

    缓冲液的pH 值对蛋白质的特性有极大的影响。一般来说，蛋白质的溶解性在它等电点时是最低的。因此开发者为了尽可能的降低溶液中的蛋白质分子的稳定性，缓冲液中最理想的PH 值应控制在所用的蛋白质的等电点附近。

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.

共沉淀试剂 

    在调整缓冲液时，为降低溶液中蛋白质分子的稳定性，开发者可以选择加入去稳定试剂沉淀剂。加入沉淀剂的作用是依赖于IgG分子的Fc与Fab片段对加入的试剂有不同的稳定性，这就是在沉淀剂的作用下，Fc片段的稳定性下降远远快于Fab片段，Fc片段的部分去稳定作用导致了更多的疏水基因的暴露，在正常情况下这些疏水基是隐藏在蛋白质分子内部的。因此，不管哪种蛋白质与NC膜结合的机制起作用，因为沉淀剂作用引起的疏水性增强都将提高蛋白质的结合能力。

    过去最常用的沉淀剂是有机醇，因为有许多理由值得推荐。有机醇的存在能帮助湿润NC膜，减少膜可能带有的静电，并且对溶液中的蛋白质有去稳定作用。在用膜作为免疫分析中，加入3-5%的甲醇能极大地提高它的性能。

    几年前就已经知道，在ELISA实验中，使用乙醇来提高蛋白质一固相的结合性能，而现在这已经成为标准方法了。脂肪醇对NC膜结合蛋白的影响首先是在1980年知道的，当时，在profeim blotting 实验加1% 异丙醇 作为固定剂在广泛的使用。尽管一些物质如硫酸铵等有时作为沉淀剂也有很好的效果，但是它们通常不如有机醇，这些类型的试剂中，甚至是微小浓度的变化都会严惩影响蛋白质的沉淀程度，正因如此，一般情况下，多用有机醇而不用其它物质作为沉淀剂。

    综合以上各主要点显示，一般情况下，新产品的开发都是以下组合的缓冲液开始的：

    10mmol 磷酸缓冲液+3%甲醇+PH7

    尽管这种缓冲液并不是对所有的蛋白质都是最佳，但是它提供了一个开发过程极好的起始点．

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:

• Membrane type. .

• Pore size.

• Posttreatments.

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.

膜对蛋白质的结合有显著的影响

    影响膜的特性的关键因素有：

• 膜的类型

• 孔径大小

• 膜的后处理

    不同类型的膜对同一种蛋白质的结合水平存在着很大的差异，这就意味着任何一个新产品的开发都必须重新筛选膜。

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.

    膜的孔径和膜的后处理均可独立地对蛋白质的结合水平产生显著的影响。图5现示了免疫球蛋白和白蛋白对于不同孔径规格及不同厂家的膜的结合水平的比较。从一系列的数据也显示蛋白质结合水平的变化与膜的标示孔径变化之间的关系不大。从图中比较也可显示对于免疫球蛋白有最佳结合的膜不一定对白蛋白也有最佳的结合能力。因此，相关蛋白的结合水平既受膜的规格的影响，也受膜的来源的影响。

    对于有固定表面积的一系列膜来说，蛋白质的结合水平是由生产膜的聚合物的类型及影响膜表面活性的处理试剂共同影响的结果，膜产品的基本聚合物都来自许多商业可用的来源，但每一个来源在特性上都有轻微的不同，而且，不同的膜生产厂家对膜都有不同的处理方法。对产品开发来说，总是建议通过实验来评价相关蛋白与他们即将打算使用的膜的结合特性。

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.

    产品开发者可用厂家标示的孔径来区分不同的膜，但应注意仅限对同一个生产厂家的产品而言。因而不推荐用标示的孔径来区别不同的厂家生产的膜材料。一般来说，标示的孔径没有一套标准方法来确定蛋白质的结合量，特别是在lateral-flow分析中，因此，开发者在开发一个新产品时，建议最好筛选一下膜的孔径范围。对于任何范围内的NC膜，膜结合蛋白质的量都将随着孔径的减小而上升，这是因为膜上实际可用的表面积增大的缘故。

    对于每一种不同孔径的膜，它大约的表面积可通过看表面积比率来确定。（SAR估算出来，SAR代表该孔径的膜实际可用的表面积与所用膜平面积的比率 <见表Ⅰ>）。还有一主要的现象是，随膜孔径的减小，膜层析率也将变小<见表Ⅱ>,层析速度减小将提高检测的灵敏度,因为被测试剂将在捕获线停留更长的时间。

    综合以上两种现象可以看出，使用膜的孔径越小，可得到的相对灵敏度就越高．因此，作为总的指导方针，若开发者最关心的是分析的最终灵敏度，那么就应选择尽可能小孔径的膜；若开发者道德考虑的是分析的速度，那么就应该选择较大孔径的膜。总之，无论需要的是什么，对开发者来说，在产品开发的早期，通一等系列的实验评价，他们总能找到一种最佳孔径的膜．

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.

    NC膜的生产的一个例行过程是膜的后处理，目的是去掉膜表面的没有聚合的小分子前体或者修饰膜的重湿润性能。在另外的一些情况下，通过这样一些的后处理过程，也可引入一些膜本身没有的痕量物质或者别的一些物质，以改良测试分析的最终结果。

    在一般情况下，生产者应该知道在膜上需加附加一些什么物质，它们的浓度是多少以及怎样来测量它们的浓度水平。通过这些附加上的物质的作用，可以看到它们对蛋白质与膜结合的水平的影响，膜的层析速率以及对膜的寿命的影响。

    在大规模的工业生产中，为保持NC膜的显润特性，膜的后处理通常来用一些实践的经验，但是，对于膜在到达客户手里之前及在试剂盒生产中点样T线后，是否进行后处理，仍然存在争论。从商家买来已经处理过的膜可能是一种很好的选择，因为这样的话就可减少试剂盒生产者额外的工作量。这些经处理过膜可以直接拿来到生产中使用，因此，这样就节约了生产设备和生产成本以及生产过程所用的时间。当然，产品开发者在决定是否接受这种已处理过的膜之前，应该考虑到以下一些能引起某些应用中不合适的问题。

    NC膜在点样捕获线之前进行后处理的一个主要缺点是湿润剂在膜上会移动可漏去，若膜被长期贮存后这种现象将更为明显。膜上湿润剂浓度的改变会影响蛋白质与膜的结合特性，当然也会影响湿润特性及层析速率。这样，产品的货架寿命将取决于用于膜的湿润剂的浓度、实际产品的可能有效期及产品在使用中某些可能的未知因素。另一方面，若生产者在购买后在内部做膜的后处理工作，那么，应记录好膜上湿润的试剂浓度及做好它们在膜上老化的研究工作。这样开发者就能够创建一个适当的后处理程序来确保膜的使用和长时间保存。

    未处理过的NC膜，其亲水性是与它的孔径直接相关。

    对于未处理的NC膜，它的亲水性是其孔径结构的直接功能。但是经过亲水性试剂处理过的膜，其亲水特性就由其后处理试剂来决定了。如果膜在贮存过程中发生后处理试剂漂移或者在测试时被样本冲走，那么膜的实际特性与它原有的特性将相比将发生很大的变化。在原始的QC检验中，能使生产者判断亲水性后处理和孔径结构所引起的对膜的复杂影响。但是若后处理试剂老化或者膜的特性发生改变，从而使得膜的特性取决于膜的结构的程度上升了，则原始的QC检验就无效了。若生产中不同批号产品使用的膜孔径结构一致，而且未经亲水试剂处理过，则能确保产品的可变性更小减小。

    因为许多后处理试剂都是水溶性的，当捕获线被测试时，任何水的存在均可把捕获线区域上的后处理试剂冲走。结果导致膜上部分位置没有湿润剂，因而高度疏水，使得被点在捕获线上的试剂接近不了被测样品中的目标试剂和金标，这将严重影响试剂盒的可读性。常常有这样的情况出现：疏水导致样品不均一通过捕获线，使捕获线结果产生条纹或显色深浅不一致。更有甚者，还在捕获线上出现反白现象。如果在点样捕获线后进行后处理，就可避免这些缺点。

    产品开发者在权衡是否使用已经后处理的原料来保持一致性还是用没有包含表面活性处理的原料来保持稳定性，应该从时间及成本方面来考虑。

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.

References

1. MA Harvey, Optimization of Nitrocellulose Membrane Based Immunoassays (Keene, NH: Schleicher & Schuell, 1991).

2. Guide to Building Molecular and Immunodiagnostic Device Platforms (Keene, NH: Schleicher & Schuell, 1997).

3. Short Guide for Developing Immunochromatographic Test Strips (Bedford, MA: Millipore Corp., 1996).

4. KD Jones, Technical Application Notes, no. 1–3 (Maidstone, UK: Whatman International, 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, 1988).

7. C Wallis, JL Melnick, and CP Gerba, "Concentration of Viruses from Water by Membrane Chromatography," Annual Review of Microbiology 33 (1979): 413–437.

8. WG Presswood, Membrane Filtration: Applications and Problems, (New York: Marcel Dekker, 1981).

9. SR Farrah, DO Shah, and LO Ingram, "Effects of Chaotropic and Antichaotropic Agents on Elution of Poliovirus Adsorbed on Membrane Filters," Proceedings of the National Academy of Science USA 78 (1981): 1229–1232.

10. B Batteiger, V Newhall, and RB Jones, "The Use of Tween 20 as a Blocking Agent in the Immunological Detection of Proteins Transferred to Nitrocellulose Membranes," Journal of Immunology Methods 55 (1982): 297–307.

11. P Tijssen, Practice and Theory of Immunoassays, 8th ed, (Amsterdam,: The Netherlands Elsevier, 1993).

12. HC Wood and TG Wreghitt, "Techniques," in ELISA in the Clinical Microbiological Laboratory, ed TG Wreghitt and P Morgan-Capner (London: PHLS, 1990), pp 6–21.

13. Z Schneider, "Aliphatic Alcohols Improve the Adsorptive Performance of Cellulose Nitrate Membranes—Application in Chromatography and Enzyme Assays," Annals of Biochemistry 108 (1980): 96–103.

14. R Bowen, private communication with author, Swansea, UK, February 3, 1998.

15. Technical Data: Nitrocellulose Membranes (Maidstone, UK: Whatman International, 1996).

16. KD Jones and AK Hopkins, "Protein Binding in Nitrocellulose Membranes 0.2 to 12 µm: A 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).