Superior stability, sensitivity, and precision and reproducibility of manufacture make gold suitable for use in membrane-based tests.

The demand for rapid membrane-based tests encompasses applications in a wide variety of fields (see box, below). The number of such potential applications will likely increase as these low-cost alternatives to expensive instrumented methods of testing become more sensitive and more specific.

    对快速诊断的需求正渗透到很多领域的应用当中（见下图）。随着较那些昂贵的检测方法廉价的检测的灵敏度和特异性的提高，这些潜在的应用数目很可能会增加。

This article focuses on the importance of a critical component of such tests—the detection label—and how its manufacture can affect the performance and reliability of the whole test system. Specifically, the article will address the advantages of using gold conjugates in this capacity.

    这篇文章主要讲述这些检测中重要物质——探测标记物的重要性，和它的制备如何影响整个检测系统的性能和可靠性。文章还特别阐述了在生产中用金标记物的 优点。

What Is a Rapid Test?

A rapid test is an inexpensive, disposable, membrane-based assay that provides visual evidence of the presence of an analyte in a liquid sample. Such tests can be formatted either as freestanding dipsticks or as devices enclosed within plastic housings. Typically, as little as 200 µl of liquid sample is required to perform the test, which is usually complete within 2–5 minutes. In clinical assays, the sample may be urine, blood, serum, saliva, or other body fluids. In nonclinical tests, the sample may be a small volume of solution prepared from soil, dust, plants, or food, and similarly applied directly to the membrane test strip. No instrumentation is required to perform such tests, which can be used in clinics, laboratories, field locations, and the home—often by inexperienced personnel.

什么是快速检测？

    快速检测是一种廉价的、可任意使用的、基于膜的检测方法，它能提供分析物存在于液体样品的视觉证明。这种检测可以以独立的试纸条，也可以以装在塑料盒中的形式进行。一般地，进行检测仅需要200ul的液体样品，一般在2－5分钟内完成。在临床检测中，样品可能有尿、血液、血清、唾液或其他体液。在非临床检测中，样品可能有由土壤、灰尘、植物或食物等准备好的小量的溶液，类似的直接应用在膜试纸条上。这种检测不需要任何仪器，能被用于临床、实验室、室外和家庭——经常由无经验的人员检测。

A large flask of gold conjugate. Photo by Hugh Burden, courtesy BB International
　

Rapid tests come in two forms—lateral flow and flow-through. The lateral-flow format is by far the most common because it is easier to manufacture and use. Although the same principles apply to both formats, the lateral-flow test is discussed here.

    快速检测分为两类——侧向流动和渗透。因为侧向流动易于生产和使用，所以它很普遍的存在。虽然两种形式的原理一样，但这里讨论的是侧向流动检测。

The base substrate of a rapid test is typically a nitrocellulose strip onto which is immobilized a capture binding protein, usually an antibody or antigen (see Figure 1). A pad (often glass fiber) containing dried conjugate is attached to the membrane strip. For the majority of currently available tests, this conjugate pad contains gold particles adsorbed with antibodies or antigens specific to the analyte being detected. A sample pad, usually paper, is attached to the conjugate pad. When applied to the sample pad, the liquid sample migrates by capillary diffusion through the conjugate pad, rehydrating the gold conjugate and allowing the interaction of the sample analyte with the conjugate. The complex of gold conjugate and analyte then moves onto the membrane strip and migrates towards the capture binding protein, where it becomes immobilized and produces a distinct signal in the form of a sharp red line. A second line, a control, may also be formed on the membrane by excess gold conjugate, indicating the test is complete.

    一个快速检测的底层一般是硝酸纤维素膜，在它上面固定了检测蛋白，通常是抗体或抗原（图1）。连接着膜的是包含有干燥金标的金标垫（通常是玻璃纤维）。在现有的大部分试纸条中，金标垫的金颗粒被能特异性作用于检测物的抗体或抗原吸附。样品垫通常是 纸，粘贴着金标垫。当液体样品用在样品垫上，液体由毛细作用力扩散通过金标垫，使金标复水并使得样品中的分析物特异性作用于金标。金标和分析物的复合物扩散到膜上，并向检测蛋白移动，在那里复合物被固定住，产生清楚的信号——一条红线。第二条线是控制线，在膜上由多余的金标记显色形成，用于表示检测完成。
　

Figure 1. Construction of a lateral-flow rapid test.

By definition, rapid tests should provide results in a short time, preferably minutes. Such tests must be convenient, accurate, reliable, inexpensive, disposable, and foolproof. They must also be easily and unambiguously interpreted, even by users without experience. From the manufacturers' point of view they should carry a large added value and be easily marketed worldwide to users who may be either experienced or inexperienced in the use of such tests. (see sidebar, below).

Rapid tests are versatile. By switching the antibodies and making small adjustments to the chemistry of the strip format, the same test design can be used for many applications.

   由定义可以看出，快速检测能在很短的时间内给出结果，准确的说是在几分钟内。这些检测必须是方便的、准确的、可靠的、廉价的、可任意使用的以及十分简单的。它们也必须容易和清楚的被说明，甚至是没有经验的使用者。从生产商的角度看，它们应该拥有大量附加价值，并且容易在全球销售给使用者，使用者可以是在试剂使用方面有经验的或没有经验的人员。

    快速检测是通用的，更换试纸条上的抗体并稍微调节其化学性质，同样的试纸条设计就能用于很多应用领域。

Why Use Gold?

Early rapid tests used colored latex to form the visual signal, and some current versions continue to use this method. Latex was originally, and still is, the prime labeling method used in agglutination tests. This is because of its predisposition to agglutinate in the presence of binding components. For rapid tests, in which stability of the conjugate is critical for avoiding false positives, this predisposition to agglutinate can become a major problem.

Because of their greater potential stability, gold labels were introduced into membrane-based rapid tests in the late 1980s. Gold particles of any accurately defined size can be manufactured reproducibly under the appropriate manufacturing conditions. Different sizes may be used for different applications. Its superior stability, sensitivity, and precision and reproducibility of manufacture make gold suitable for use in rapid tests. Gold is essentially inert and forms almost perfectly spherical particles when properly manufactured. Proteins bind to the surfaces of these gold particles with enormous strength when correctly coupled, thus providing a high degree of long-term stability in both liquid and dried forms. Also, when accurately stabilized during manufacture, nonspecific interaction of gold conjugates can be reduced to zero.

使用金标记的原因

    在初期，快速诊断用乳胶作为标记物来产生信号，现在仍有一些产品使用这个方法。过去和现在，乳胶法一直是凝集反应主要的标记方法，这是因为它在结合成分的存在下易于凝集。对于快速诊断试剂来说，标记物的稳定性对避免假阳性是非常重要的，因此易凝集的特性会成为大难题。

    因为具有强潜在稳定性，金标记在80年代后期被引入到快速诊断试剂中。在合适的生产条件下，任何精确大小的金颗粒都可以生产。不同的大小有不同的应用。生产的高稳定性、高灵敏度、高精度和高可重复性使得金适合用于在快速诊断试剂中。如正确制造，金是惰性的球型颗粒。当正确连接时，蛋白质能牢固地结合在金颗粒表面，无论是液态或固态都能保持长久的稳定性。稳定生产能使金标的非特异性结合为零。

A comparison of the benefits of different labels used to mark antibodies and antigens is shown in Table I.

Table I. Comparison of the characteristics of labels commonly used in rapid tests.

Manufacture of Gold Colloids

When producing large volumes of gold conjugate, sophisticated processing methods are required to attain reproducible batch-to-batch manufacture and to avoid instability. The manufacture of 100 liters of high-quality gold conjugate requires the utmost care and attention in order to achieve a final stable and sensitive product. Electron microscopy must be used to provide quality control at each step of the manufacture.

During the past 20 years, manufacturers have introduced a variety of different methods for synthesizing gold colloids. The goal has been to obtain colloids of a monodisperse nature, which are of a controlled and uniform diameter. Although all of the production methods rely upon the reduction of tetrachloric acid (HAuCl4) to form gold atoms, they vary considerably in the physical conditions, order of reagent addition, reducing agent used, and quality of the final colloid produced (size, shape, and coefficient of variance).

In general, all of the production methods use a reducer to donate electrons to the positively charged gold ions in solution and produce atomic gold. This is shown as follows:

Gold tetrachloric acid + reducer = gold colloid

HAuCl₄ + e– = Au⁰

Commonly used reducers include sodium citrate, yellow phosphorus, sodium borohydride, and sodium thiocyanate. Figure 2 shows the process of reduction at the ionic level.

Figure 2. Reduction of gold ions to form gold particles.

Before the addition of reducer, there are 100% gold ions in solution. The ordinate of the graph indicates the progress from gold ions to gold atoms as the reducer is added. Immediately after the reducer is added, there is a sharp rise in gold atom content in the solution until this level reaches supersaturation. Aggregation then occurs, in a process called nucleation, to form central icosahedral gold cores of 11 atoms at nucleation sites. The formation of nucleation sites, in order to reduce the supersaturation of gold atoms in solution, occurs extremely quickly. Once this is achieved, the remaining gold atoms in solution continue to bind to the nucleation sites under an energy-reducing gradient until all atoms are removed from solution.

The number of nuclei formed initially determines how many particles finally grow in the solution. This number, in turn, depends on the amount of reducer added. A large amount of reducer produces a large number of nucleation sites and hence a large number of gold particles. Clearly, the larger the number of nucleation sites for a given amount of gold chloride in solution, the smaller will be the final size of each gold particle. Particle size is thus carefully controlled by the amount of reducer added. If manufacturing conditions are optimized, then all nucleation sites will be formed instantaneously and simultaneously, resulting in all gold particles growing to exactly the same size (monodispersal). This is very difficult to do. Most manufacturing methods do not achieve instantaneous reduction and formation of nucleation sites, resulting in uneven growth and a multidisperse colloid that is virtually unreproducible and results in a very unstable conjugate.

A gold colloid comprises a suspension of gold particles individually surrounded by a negative charge layer arising from the residual negative ions in solution (see Figure 3). This charge layer, called the zeta potential, provides the means for the gold particles to repel one another and to stay in suspension indefinitely. The zeta potential can be compressed or expanded depending on the total ionic concentration of the surrounding solution.

Figure 3. Colloidal gold particle surrounded by a double ionic layer.

High-quality gold colloids and custom-manufactured conjugates are readily available commercially. By purchasing these components from a reputable supplier—instead of attempting to process large volumes of gold themselves—manufacturers can save time and considerably reduce their manufacturing risks.

胶体金的制备

    当生产大批量金标记物时，为保证批间重复性及避免不稳定性，成熟的工艺方法是必须的。生产100升高质量的金标需要非常小心以获得最终稳定及灵敏的产品。电子显微镜用于控制生产中每一阶段的质量。

    在过去20年中，生产者提出了多种不同方法来合成胶体金，目的是为了得到单分散性胶体金—受控的均一的胶体金。虽然所有的生产方法都是还原HAuCl4形成金原子，但是它们还受以下条件影响：物理条件、试剂的添加顺序、使用的还原剂和最终胶体的质量（大小、形状和变异系数）。

    一般来说，所有的生产方法是使用还原剂提供电子给溶液中的正价金离子而生成金原子，反应公式如下：

                                             HAuCl4 + e- =Au0

    通常使用的还原剂包括柠檬酸钠、黄磷、硼氢化纳、硫氰酸钠。

    在加还原剂之前,溶液中金离子是100%，曲线图的纵坐标表示加入还原剂后从金离子变为金原子的过程。加入还原剂后短时间内，溶液中的金原子含量直线上升直到饱和。开始出现聚集，这个过程叫做成核现象，形成11个原子构成的中央二十面体金核。为减少溶液中过饱和的金原子，晶核构架形成得非常块。一旦晶核形成，溶液中剩余的金原子依据能量递减梯度继续结合到晶核上直到所有原子从溶液中消失。

    最初形成晶核的数量决定最终溶液中有多少金颗粒。颗粒数量是由加入还原剂的量决定的。还原剂越多产生的晶核越多从而产生的金颗粒也越多。很明显，大量晶核必然导致金颗粒变小。因此，金颗粒的大小由加入还原剂的量多少控制。如果优化生产条件，所有的晶核将在瞬间同时形成，导致金颗粒达到均一的大小（单分散性）。但这是很难做到的。大部分的生产方法不能瞬间还原形成晶核，从而导致不均匀聚集，多分散性的胶体没有可重复性，导致形成非常不稳定的金标。

    胶体金由金颗粒悬浮液构成，金颗粒被一个负电荷层包被着（见图）。这个电荷层称作zeta电势，它能使金颗粒互斥并在悬浮液中任意分布。zeta电势能随溶液中的离子浓度收缩或扩张。

    高质量的胶体金和为客户生产的金标在市场上随处可见。购买知名供应商的原料——代替自行生产大批量胶体金——能节约时间并降低生产风险。

Good Gold, Bad Gold

The apparent ease with which gold colloids and conjugates can be manufactured has led to the commercial availability of many poor-quality, poorly characterized, and nonreproducible products. When incorporated into rapid tests, such products can lead to poor stability, sensitivity, and specificity. To prevent this, gold colloids should be evaluated ultrastructurally using a transmission electron microscope (TEM). Such an evaluation should enable the manufacturer to compare the diameter of the colloids to that of a calibrated standard and to obtain information about particle sphericity, particle irregularity, and overall variance in particle diameters.

Unevenly shaped particles occur when the nucleation and growth rate during the reduction process have been uncontrolled (see Figure 4). Particles may be seen to be of different sizes and shapes (oval, triangular, oblong, rhomboid, etc.). These particles will not coat evenly with protein during conjugation and will not evenly repel one another in solution. This can affect the color, sensitivity, specificity, and stability of the final product. A mere 5% of odd-shaped particles can influence a test result, making it completely nonreproducible. The signal formed by such "bad gold" is usually seen as having a bluish or purple color compared with the normal cherry red that typifies a well made 40-nm monodisperse colloid. Although the darker color may be easier to see against the white membrane of a test system, it indicates an inherent instability that is much more likely to give false results. Even worse, uneven particle shape and size produce very uneven protein coating, which leads to long-term clustering and aggregation of the conjugate. Such changes may occur within days or even hours of storage of the conjugate in solution.

Figure 4. Good gold colloids and bad (unstable) gold colloids.

Even drying the conjugate immediately onto the solid-phase substrate does not entirely overcome this problem. During the drying process, surface proteins that are not securely attached because of particle shapes, can easily become detached, yielding false negatives and high background.

One of the most common problems encountered in rapid-test performance is failure of the gold conjugate to release at the correct speed and with integrity from the glass-fiber conjugate pad. This failing is often the result of poorly coated gold particles being directly exposed to the glass-fiber material and not being able to release. This inevitably means that surface proteins have either become permanently attached to the fiber matrix or have left the gold particles and are floating free. With well-prepared gold conjugates, starting with evenly coated monodisperse and spherical particles, such dangers are greatly reduced.

Although TEM examination is the only true way to determine the quality of colloid, a quick method for determining whether a colloid or conjugate contains fused, aggregated, or heterogeneous particles, or a mixed-size population, is to examine its color visually. A good 40-nm colloid (the most frequently used size for diagnostic applications) should be cherry red. If the colloid or conjugate appears purple, it is likely to be of poor quality and unstable.

好胶体金和不好的胶体金

    胶体金和金标能在明显的简单条件下制备出来，可是简单的条件已经导致市面上的低质量、差特性和无可重复性的产品。当装配到快诊试剂上时，这种产品的稳定性、灵敏度和特异性都很差。为了防止这个问题，可用电子显微镜来检测胶体金的超细微结构。这种检测能使生产者对照标准比较胶体的直径，得到颗粒球状、颗粒不规则性和颗粒直径的变化。

    当还原过程中结晶和聚集速度不能控制时，形状不规则的颗粒将会产生（见图4）。颗粒呈现不同的大小和形状（椭圆形、三角形、长方形、斜长方形等等）。这些颗粒在标记时表面不能均匀包被蛋白质，也不能在溶液中均匀分布，这会影响成品的颜色、灵敏度、特异性和稳定性。仅仅5%的异性颗粒就会影响检验结果，使之没有可重复性。使用不好的胶体金产生的颜色是带蓝色或紫色，而用40nm单分散性胶体产生的是正常的粉红色。虽然在试剂白背景膜上深的颜色更容易看到，但暗示着潜在的不稳定性，即更可能得到错误的结果。更糟糕的是，不均一的颗粒形状和大小会产生很不均匀的蛋白质包被，会导致金标长期结块和聚合。这种变化在金标存放期的几天甚至几小时内发生。

    甚至将金标喷到膜上后快速干燥也不能彻底解决这个问题。在干燥过程中，因为颗粒形状而不能紧密结合，使得表面蛋白质很容易与胶体金分离，产生假阴性和深背景。

    在快诊试剂检测中经常遇到的问题之一是金标不能以正常速度并且完全地从金标垫上释放。这个问题常常是因为未完全包被的金颗粒直接暴露在垫上而不能释放。这就意味着金表面的蛋白质或永远吸附在基材上，或脱离金颗粒被冲走了。使用精制的金标，即使用均一包被的单分散球形颗粒，这些问题都会明显减少。

    虽然电镜检查是检验胶体金质量的唯一正确的方法，但有还有一种快速检验方法是目测它的颜色，它能用来检测胶体金或金标是否含有聚集的、融合的或异类的或混合大小的颗粒。一个好的40nm的胶体金（诊断试剂最常用的尺寸）应该是粉红色。如果胶体或金标呈现紫色，可能是低质量的、不稳定的。

Choice of Gold Particle Size

The signal is generated on the test strip by the accumulation of gold particles at the test or control line. These particles must be large enough to be seen. The greater the particle size, the easier it is to see an accumulation of these particles. For example, particles of 1 nm diameter would be virtually impossible to see, no matter how many had accumulated, because 1-nm particles do not have the bright red color of the larger sizes. It is not until particles reach 20 nm that a worthwhile signal can be seen. Steric hindrance becomes a problem as the particles increase in size (see Figure 5). For example, if an IgG molecule (160,000 daltons) is just 8 nm in length, only approximately 4 nm of this will extend from the surface of a gold particle. Particles of 100 nm will tend to dwarf the small surface molecules and make it difficult for them to interact with specific proteins. In addition, the larger the particle size, the fewer of them can be contained in a given volume of solution.

Figure 5. Choice of gold particle size for optimized signal.

This trade-off between required visibility and steric hindrance dictates that, for most immunoassay applications, the optimum particle size is 40 nm. In some cases where steric hindrance is a greater problem (e.g., for smaller antigens), particles of 20 nm are preferred. Larger particles may be preferred when a darker red color is desired or when the lower curvature of the particle surface would improve the molecular interaction between the antibody and antigen. Experiments should be performed to determine which particle size gives the highest sensitivity with the lowest background and greatest stability.

金颗粒大小的选择

    试剂条上信号的产生是因为金颗粒在C、T线的积累。这些颗粒必须大到能够被看到。颗粒越大，颗粒的积累越容易看到。例如，直径1nm的颗粒不管积累多少也不能被看到，因为1nm颗粒不具备大颗粒的粉红色。直到颗粒增大到20nm标记才可见。当颗粒增大时，空间障碍又成为一个问题（见图5）。例如，一个IgG分子（160,000daltons）在长度上只有8nm，大约4nm从金颗粒表面伸展开。100nm的颗粒有使表面分子变小的趋势，使它们难和特异蛋白结合。另外，颗粒越大，一定体积溶液中所含的数量越少。

    可见性和空间障碍的平衡如图，对多数免疫试剂应用来说，最佳的颗粒大小是40nm . 在一些情况下，空间障碍是一个大问题（如小抗原），20nm的颗粒更好。当需要暗红色或颗粒表面较小的曲率能增加抗原抗体间的分子引力时，大颗粒更好。应该由实验决定确定多少颗粒大小能带来高灵敏度、浅背景色和高稳定性。

Manufacturing Gold Conjugates

Manufacturers should understand the chemical and physical processes that permit a permanent coupling of their protein to the gold colloid. Armed with this knowledge, it is then simple to proceed from small test batches to large-scale manufacturing without compromising sensitivity, stability, specificity, or reproducibility. Simply throwing a conjugate together, while cheap and easy, will often lead to the formation of clusters. Viewed through a TEM, each cluster is a group of four or more gold conjugate particles. The presence of such clusters generally indicates an unstable product whose performance will change over time. Although initially appearing more sensitive than an unclustered product, such tests will soon begin to show stability and specificity problems.

A good conjugate is one where the protein is adsorbed onto the surface of the gold. Unlike latex, proteins are only adsorbed passively onto the surface; covalent coupling is not performed. However, hormones, drugs, and other small molecules (<10 kD) must first be coupled to a larger carrier protein, typically BSA, before passive conjugation can be performed. This is because there are not enough points of contact between the molecule and the gold surface. Steric hindrance would in any case prohibit the small molecule from rising above the zeta potential. The carrier should be inert, in that it should have no effect on detector protein activity.

Figure 6. Binding forces between an antibody and a gold particle.

The antibody will be firmly attached to the gold by the Fc region, leaving the Fab region protruding through the double-ionic layer surrounding the gold particle and able to bind the analyte (see Figure 6). There must be no excess of labeling antibody present. Such excess will cause unlabeled antibody to compete with the labeled antibody, resulting in the presence of false negatives, and may affect the long-term stability of the conjugate. Optimum binding of proteins to gold occurs at, or close to, the isoelectric point.

Adsorption of protein to gold occurs within a matter of seconds through the follwing mechanisms:

·    Initial charge attraction of the negative gold particle to the positively charged amino acids within the protein (e.g., lysine).

·    Hydrophobic adsorption of the protein to the particle surface, through certain amino acid residues including tryptophan.

·     Dative binding between sulphur residues on the protein (from cysteine residues) and the gold particle.

金标的制备

    生产者应清楚蛋白质永久结合到胶体金上的化学和物理过程。具备了这些知识，很容易从小批量生产过渡到大批量，而不牺牲灵敏度、稳定性、特异性或可重复性。仅仅将金标简单的混合在一起，既便宜又容易的，然而常常会导致结块。通过电镜观察，每个结块是四个或更多个金颗粒的聚合体。这些结块的存在意味着不稳定的产品性能会随时间改变。虽然比较未结块的产品刚开始具有更高的灵敏度，但这些产品很快会出现稳定性和特异性的问题。

    一个好的金标就是蛋白质吸附在金的表面。和乳胶不同，蛋白质只是被动吸附在表面，并没有共价键。然而，激素、毒品和其它一些小分子（<10kD）在被动结合前必须先和大分子载体偶合（最典型的是BSA）。这是因为在分子和金表面没有足够的连接点。空间障碍会阻碍小分子在金的zeta电位上出现。载体应该是惰性的，这样就不会影响检测蛋白质的活性。

    抗体的Fc片段紧紧吸附在金颗粒表面上，这样围绕着金颗粒的双电子层表面的Fab片段就突出在外面，这样Fab就能吸附分析物（见图6）。胶体金上标记抗体时，不能有过量的待标记抗体存在。因为过量的游离的抗体会跟胶体金上的抗体与抗原发生竞争反应，导致出现假阴性，而且过量的抗体蛋白会影响到金标的稳定性。蛋白质和胶体金结合的最佳条件在蛋白质的等电点附近。

Stabilizing the Conjugate

Following the conjugation of the specific protein, the conjugate must then be stabilized with a suitable agent. BSA, gelatin, PEG (Carbowax), or casein are commonly used. The purpose of the stabilizer is twofold. First, it reduces nonspecific interactions by blocking any sites on the colloidal surface that are not occupied by the specific protein. Second, it helps provide a more-stable suspension.

Once conjugated, the gold is adjusted to the desired concentration, and suspended in a suitable buffer. This buffer should confer stability to the liquid conjugate. Commercial manufacturers will often use a low-molarity buffer so that the consumer can easily resuspend the conjugate in the buffer of his or her choice. Generally, high-salt buffers and surfactants should be omitted from the final storage buffer as they may cause damage by hydrolysis or displacement of the antibody. Preservatives containing sulphur residues or mercury will cause the conjugate to collapse. The most common preservative used for gold conjugates is 0.1% sodium azide.

Expertly manufactured gold colloid has a virtually infinite shelf life. By calculating how much binding agent can be adsorbed onto the surfaces of the gold particles, it is possible to minimize batch-to-batch variation, and the resulting conjugate has a longer shelf life than antibody alone. Correctly dried onto solid-phase components, these gold-labeled proteins remain stable for many years.

稳定金标记物

    在和特异蛋白结合后，金标必须用适当的试剂进行稳定。通常使用BSA、凝胶、聚乙二醇或酪蛋白。稳定剂的目的是双重的，第一，它通过封闭胶体金表面未和特异性蛋白结合的位点而减少了非特异性反应；第二，它提供了一种更稳定的悬浮液。

    一旦标记上，金标要被调整到所需的浓度，悬浮于适当的缓冲液中。这种缓冲液应能使液体金标稳定。最终储存用的缓冲液应避免使用高盐缓冲液和表面活性剂，因为它们可能水解或取代抗体。含有硫基或贡的防腐剂会使标记崩溃。金标最常用的防腐剂使0.1%的叠氮钠。

    熟练生产的金标有效期很长。通过计算有多少抗体能吸附在金颗粒表面，可能会减少批间差，金标也会比抗体单独储存有更长的有效期。正确地干燥金标，会使得这些金标记蛋白质能保存很多年。

Manufacturing Bulk Quantities of Gold Conjugates

Although technical and scientific sources describe the simple small-volume manufacture of gold colloids and conjugates, the commercial methods used for large-volume manufacture (i.e., 100-liter batches) remain proprietary. Given that the reduction process of gold chloride to gold colloid must take place in microseconds, the accomplishment of this in volumes of 100 liters or more requires very sophisticated techniques that do not follow the same rules as small-volume production.

Because these techniques are known only to specialists in the gold conjugate industry, whose products are commercially available, many manufacturers choose not to attempt the difficult task of producing large volumes of gold colloids in-house. Instead, such rapid-test manufacturers are partnering with gold specialists, thereby reducing their overhead costs and risk of failure, while also making it possible to get their product to market as quickly as possible.

Such partnership arrangements typically begin with the manufacturer conducting in-house experiments using small volumes of commercially available colloids in order to determine which antibodies are most suitable for their application. Following that, some in-house conjugation experiments using larger volumes of colloids are conducted to determine whether scaling up by 10x and by 100x produces the same quality of conjugate. It is important at this stage to perform rigorous stability trials of the gold conjugate at elevated temperatures and in a variety of buffers as well as in the dried-down state. Quality control should include checks on clustering (by TEM), optical density, sensitivity, and specificity. Several batches should be made at these higher volumes to determine the reproducibility of manufacture before committing to rapid-test batch production.

生产大批量的金标

    虽然有技术和科学资料描述了小批量制备胶体金和金标的方法，但是用于大批量制备（100升批量）的商业方法还是由个别企业拥有。假设由氯化金生成胶体金的还原过程必须在微秒中完成，那么要在100L体积中完成更需要熟练的技术，这和在小批量生产的标准是不一样的。

    因为只有金标行业中的专家才拥有这些技术，他们的产品都已经商业化，所以很多厂家选择不尝试自行生产大批量胶体金的艰难工作。相反，这些快速检测厂家和胶体金专家合作，因而减少他们的高费用和失败的风险，也能使得他们的产品能够更快的上市。

    这种关系的建立，通常刚开始厂家用少量的商业胶体金来实验，来确定哪种抗体最适于应用。接着，一些实验室内的结合实验用大量的胶体金来检测放大10倍和放大100倍能不能得到相同的结合物质量。在这个阶段，在高温度下、各种缓冲体系和向下干燥状态下金结合的严格稳定性实验是很重要的。质量控制包括检测聚集（用TEM）、光密度、灵敏度和特异性。在用于快速检测批次生产前，应该在这些较大体积下分批生产若干，来确定生产制备的可重复性。

Possibilities for Quantitation

Most rapid tests are purely qualitative, designed only to indicate whether a particular biochemical component is present in the clinical sample above a detectable level. However, many other tests require an element of quantitative measurement, such as for monitoring the progression of a clinical condition or for determining the relative or absolute levels of particular analytes.

Such tests fall into three types. In the first, a number of built-in signals allow visual comparison of test results as the test is being performed. This type of test can provide only semiquantitative results, where the signal may fall into a preestablished negative-, low-, medium-, or high-result category.

In the second type of test, lines of capture reagent in the test zone are set to capture only a known quantity of analyte. Any excess analyte is captured by subsequent capture lines, thus producing a ladder of signal lines that exhibit a thermometer-type display on the strip.

In a third type of test, the signal generated by the test sample is read by a small, portable analyzer that converts the colored test line to a digital signal. A number of such generic test-strip readers have recently become commercially available.

Gold labels are especially suited to such quantitative or semiquantitative tests because the signals they generate are both accurate and highly reproducible. Signals are most often read by reflectance, with photodiodes producing numbers against a calibrated scale. Precision depends on both how accurately the test result can be read, and how reliably the test strip generates a reproducible signal. Because of the precision of gold labels, the latter can be achieved with greater confidence than with many other types of label.

定量的可行性

    大部分的快速诊断试剂是定性的，只能用于检测临床样本中是否含有超过检测浓度的特定生物成分存在。然而很多其它检测需要定量，例如监控临床指标的变化或确定特定分析物的相对或绝对浓度。

    这种定量检测分为3种类型。第一种通过试纸条的颜色深浅的视觉比较，这种检测只能提供半定量结果，结果可分为阴性、弱阳性、中阳性和强阳性。

    第二种类型，检测线的反应剂设计成只能结合已知一定量的分析物，任何过量的分析物将会和下一条检测线结合，产生一个温度计式的条带梯度。

    第三种用便携式的READER来读产生的条带，将颜色浓度转化为数字指标。

    金标非常适合用于这种定量或半定量的检测因为产生的条带既精确又有高可重复性。条带大多可用反射原理读到，光电二极管产生一个经过校正的数值。精度取决于读取结果的精确性和试剂条的可重复性。因为金标的精度高，能取得比其它标记更好的结果。

Silver Enhancement of Gold Signals

Whether in the lateral-flow or flow-through format, rapid tests commonly offer sensitivities similar to or greater than ELISA kits. For many analytes, it is possible to achieve detection levels of 1 ng/ml or less. There are still many analytes, however, for which this direct-labeling system does not produce great enough sensitivity.

Sensitivity is limited by the ability of the user to visually detect the gold signal on a white membrane against a clear background. Quantitative instrumentation using light reflectance techniques usually does not achieve any greater sensitivity than the human eye. A valuable and promising technique for obtaining over 100x increased detection sensitivity is the use of gold labels enhanced with silver.1 In this method, the initial (sometimes invisible) signal from the gold label is enhanced through the application of a silver solution to the test line. Where the particles used in the gold conjugate are small (~5 nm), greater overall sensitivity can be achieved by using this indirect method. Enhancement can be achieved without any extra steps by incorporating the silver reagents into the one-step test so that application of the liquid sample drives the entire reaction (see Figure 7). All reagents (both gold conjugates and silver enhancement salts) are dried onto the test strip and are solubilized by the sample alone.

Figure 7. Silver enhancement of gold signals in a lateral-flow rapid test.

This indirect enhancement technique is expected to result in analyte detection in the picogram/ml range. The dried reagents are stable and may be introduced into the test devices before assembly. Because of the increase in sensitivity, much lower sample volumes may be used than in current directly labeled test systems. With silver enhancement, it may be possible to develop one-step gold-based tests for analytes not previously detectable by rapid-test configurations.

银对金信号的增强

    不论以侧向流动或渗透的形式，快速检测的灵敏度一般比ELISA试剂盒相同或更强些。对于很多检测物，检测水平都能达到1ng/ml或更低。然而仍然存在很多分析物，用于检测它们的直接标记系统还不能提供足够的灵敏度。

    灵敏度受到使用者对照空白背景视觉检测金信号的能力。使用光反射技术的定量仪器达到的灵敏度一般也没有比人的眼睛高。一个颇有价值和潜力的技术，能提高灵敏度将近100倍，就是使用银来提高信号。在这种方法中，金标的最初的信号通过银溶液应用在检测线上而得到提高。在这里，用于金标的金颗粒一般都很小（5nm左右），使用这种间接的方法能够大幅度的提高灵敏度。这种提高不需要其他多余的步骤，只需将银试剂合并到一步试剂中，这样加入液体样品就会推动整个反应进行。全部试剂（包括金标和银增强盐）在试纸条上干燥，并由样品溶解。

    这种间接加强技术认为能检测pg/ml水平的分析物。干燥的试剂很稳定，可以在组装前加到试剂装置中。因为增高了灵敏度，可能用到比现在的直接检测系统还少的样品体积。有了银加强技术，就可以开发以前不能快速检测的分析物的一步金检测试剂。

Conclusion

Rapid tests have changed little since their introduction in the mid-1980s, when latex was commonly used as the conjugate. But since then, the greater demands of sensitivity, reliability, and reproducibility have made gold the conjugate of choice for the vast majority of rapid-test designs.

Well-made gold reagents have tremendous stability, in both the liquid and dried forms. Only the most rigorous approach to manufacturing and using gold colloids will be successful if the range of applications for gold-based rapid tests continues to require even greater sensitivity and specificity.

结论

    当乳胶普遍用于结合物时，快速检测从1980年代中期开始没有太大的变化。但是此后，对灵敏度、可靠性和可重复性的更高要求使得金成为大多数快速检测设计的选择。

    无论以液体和干燥的形式，精制的金试剂具有极高的稳定性。如果金快速检测应用持续需要更高的灵敏度和特异性，那么只有用严格的方法来生产和使用胶体金才能成功。

Reference:

1.J Chandler, Assay device and method, 96901447.1. European patent application, British Biocell International.

2.John Chandler, PhD, is the managing director, and Tracey Gurmin and Nicola Robinson are part of the custom consultancy team at BBInternational (Cardiff, Wales, UK).