An analytical system for a one-step immunoassay has been constructed using the concept of immunochromatography.

The system employed two different antibodies that bound distinctepitopes of an analyte molecule: an antibody labeled with a signalgenerator (e.g., colloidal gold), which was placed in the dry stateat a predetermined site on a glass-fiber membrane, and anotherantibody immobilized on the surface of a nitrocellulose membrane.Three membranes, one with the tracer, one with immobilizedantibody, and a cellulose membrane as the absorbent of medium (in a sequence from the bottom), were attached to aplastic film and cut into strips. Aqueous medium containing analyteabsorbed from the bottom end of the immunostrip dissolvedthe labeled antibody, and the antigen–antibody binding complexformed was transported into the next nitrocellulose membrane bythe flow caused by capillary action. The complex subsequentlyreacted with the immobilized antibody, which generated a signal in proportion to the analyte concentration. The convective masstransfer of the immunoreactant to the binding partner allowed theassay to be performed with no handling of reagents. The reaction,however, was carried out under nonequilibrium conditions, whichresulted in decreased sensitivity as compared with assays performedin an equilibrium mode (e.g., ELISA). To minimize suchsacrifice, major factors that control system performance were identified and the system was then devised under optimal conditions.

    一个用于一步免疫检测的分析系统已经用免疫层析原理构造出来了。在这个系统中用到能结合到一个分析物分子的不同抗原决定基的两个不同的抗体：一个抗体用信号产生物（如胶体金）标记，在干燥的情况下放置在一个玻璃纤维膜上预定的位置；另一个抗体则固定在一个硝酸纤维素膜的表面。这里有三个膜粘在塑胶胶片上并割成条状，一个放有追踪物，一个固定抗体，最后一个纤维膜用于吸收介质溶液（从底部依次顺序）。含有分析物的水溶液从免疫条的底端吸收并溶解了标记抗体，然后合成的抗原－抗体复合物在毛细作用力的驱动下流到下一个硝酸纤维素膜。随后复合物将与固定化的抗体反应，产生的信号和分析物的浓度成比例。免疫反应物对流传递到固定化的抗体，这使得不需要操作反应物就能进行检测。然而，反应是在非平衡态的条件下进行的，这导致其灵敏度相比较在平衡态(如ELISA)的检测效果而言要差。为了减少这种损失，先确认控制系统性能的主要因素，然后在最优条件下设计系统。

For the past 40 years, utilization of most immunoassayshas been confined to laboratories equipped withtools and devices for analysis, and analysis has beencontrolled by skilled persons. However, instantaneousexamination of changes in one’s own physical symptomssymptomsor health status is increasingly preferred. Selftestsperformed at home may be an integral part offuture health care systems (1). In fact, the marketexpansion of home-version diagnostic kits in developedcountries, typically in the United States, far exceedsthe average for overall in vitro diagnostic products. Thefirst commercially successful kit was the pregnancytest based on the rapid detection of human chorionicgonadotropin in urine by simply adding urine to the test kit (2).

    在过去的40年里，大多数免疫检测的使用都只局限于装有用于分析的设备和仪器的实验室，而且分析都由熟练的人来控制。然而，对一个人自身的身体症状或健康状态的即时检测日益重要。在家进行自身检测可能是将来的健康保障系统中一个完整的部分。事实上，家庭诊断试剂盒在发达国家的市场扩张，以美国为代表，远远地超过了全部体外诊断产品的平均值。第一个商业化成功的试剂盒是怀孕检测，简单地将尿液加入试剂盒中就能快速地检测到尿液中的人绒毛膜促性腺激素。 

The convenience and speed of the test have beenachieved by a novel concept of immunochromatographicassay which depends on the transport of areactant to its binding partner immobilized on the surfacesof the membrane (1, 3–6). The transfer is inducedby the capillary action of aqueous medium throughmembrane pores and, hence, also separates the unboundreactant from the binding complex formed at theliquid–solid interface. Immunochromatographic assaynot only accelerates the analytical procedure but alsoprovides a means for performing the test without thehandling of reagents, i.e., allowing a one-step assay.Therefore, the assay may be carried out at the siteswhere the specimen is collected rather than at a specializedlocation.

    检测的方便和迅速是由这种靠一个反应物传输到另一个固定在膜表面的结合反应的新颖的免疫层析概念完成的。传递是由膜孔引起的水溶液的毛孔作用驱使的，并且从此还将未束缚的反应物从在液固界面形成的结合物中分离。免疫层析检测不仅加快了分析程序而且还提供了一种不用操作反应物就能进行检测的方法，也就是允许一步检测。因此，可以在样本收集地进行检测，而不用到一个专门的地点检测。

In this article, we describe methodologies needed inmanufacturing the immunoassay kit from the preparationof each component to the stabilization of thesystem constructed. Because the concentration rangeto be monitored is usually varied according to targetanalyte, we propose methods for controlling the extentof detection on the basis of experience. Alternativeapproaches overcoming potential problems that mayarise in arranging variable components for differentsystems are also provided.

    在这篇文章中，我们描述了制造免疫检测试剂盒所需的方法学，从各种成分的准备到构造成的系统的稳定。因为受监控的浓度范围是根据目标分析物的不同而不同的，所以我们建议基于经验的检测来控制范围。这里还提供了一些可选的方法，用来克服用于不同系统的安置不同成分引起的潜在问题。

DESCRIPTION OF METHOD(方法的描述)

Configurations of Immunochromatographic System

(免疫层析系统的结构)

Systems based on immunochromatography can beconstructed for both qualitative and quantitative analyses(see Fig. 1 for different configurations). Becausethe analytical system was initially developed for onsitedetermination of pregnancy (2), it has been devisedas an on/off format without adopting a signal detector.In the model in Fig. 1A, two antibodies binding distinctepitopes present on an analyte molecule are used: one(detection antibody) labeled with a signal generator(e.g., latex beads (3, 7), gold colloids (8, 9)) and theother (capture antibody) immobilized onto solid surfaces.The labeled antibody is placed in a dehydratedstate within a glass-fiber membrane such that the binder can be instantaneously dissolved on contact with an aqueous medium containing analyte; i.e., the substance to be measured. The antibody then participates in the binding reaction to form a complex with the analyte in the liquid phase. This complex moves forward continuously until it is eventually captured by the antibody immobilized on the surfaces of the nitrocellulose membrane. The membrane provides a uniform pore and, thus, a liquid–solid interface for reproducible antigen–antibody binding. An additional antibody specific to the detection antibody can be used to produce a control signal. The two membranes with immunoreagents are cut into strips and are contiguously attached in length (Fig. 1A), and a cellulose membrane is located at the top to induce a continuous wicking that enables the immune complex to be pulled to the immobilized antibody. A color signal can be read within 10 min, and the intensity determines the amount of analyte. 

    建立在免疫层析上的系统能被用于定性和定量分析。因为分析系统最初是为怀孕的现场检测发展的，所以已经设计作为一个有或无的结构，不需要用到信号检测器。Fig.1A的模型用到能结合分析物分子的不同抗原决定基的两个不同的抗体：一个（检测抗体）是标记有信号产生物（如乳胶珠、胶体金），另一个（捕获抗体）则固定在固体表面上。将标记抗体以无水状态放在玻璃纤维膜里，这样抗体一接触含有分析物的水溶液，即要检测的物质，就会自动溶解。然后抗体参与结合反应，在水溶液中形成了带有分析物的复合物。这个复合物继续前移，直到被固定在硝酸纤维素表面的抗体捕获为止。膜拥有统一的孔径，从而也就有了能用于可重复的进行抗原抗体结合的液固界面。另外的一个能特异性作用于检测抗体的抗体被用来产生控制信号。带有免疫试剂的两个膜被割成条状，在长度方向连续的粘贴上（Fig.1A），位于顶端的纤维素膜用来产生能将免疫复合物拉到固定化抗体的连续毛细作用力。颜色信号在10分钟内就能读出，亮度确定了分析物的多少。 

Alternatively, the color can be quantified by optical density using a sensor based on scanning photometry(7, 9) or charge-coupled device (CCD) (10), and, therefore, the immunostrip is also capable of assessment of analyte concentrations. This type of application may require certain modifications of the immunoassay system such that the site of signal generation be expanded to gain reproducible, sufficient increment of color in dose responses. The detection site can be allocated as a square membrane (approximately 5 3 5 mm) with the capture antibody coated on the surface (Fig. 1B). The detection antibody–analyte complex formation is enhanced by inserting a reaction pad between the glass strip and the capture membrane. We have found that the complex density was directly proportional to the length of the reaction pad as well as the number of pads even if total length was kept constant. The pad could also be used for the immobilization of antigen (i.e., analyte; refer to Fig. 1C), by separating the unreacted labeled antibody from the immune complex formed. Such action of the antigen pad in the analytical system makes the slope of the dose–response curve steeper but the sensitivity decreases. This results from the competition between the fixed and free antigen for binding to the labeled antibody. For a certain analyte that distributes within a narrow concentration range, the varied pattern of the dose–response curve might be necessary for accurate evaluation of unknown samples.   

   另外，使用基于扫描测光法的传感器或电荷连接装置（CCD），能用光密度定量颜色，因此，免疫条还能估测分析物浓度。这类应用可能需要对免疫检测系统进行某些更改，比如扩展信号产生部位来得到在剂量响应中可再生、充分颜色增量。可以在检测部位分配一个正方形的膜（大约5×5mm），在其表面上铺有捕获抗体（Fig.1B）。在玻璃条和捕获膜之间加入一个反应垫，可以增强检测物抗体抗原结合物的形成。我们已经发现结合物的浓度与反应垫的长度和反应垫的数量，如果长度不变的话，有直接的比例关系。还有能用于抗原（即分析物，参考Fig.1C）固定的衬垫，通过将未反应的标记抗体从形成的免疫结合物中分离出来实现。抗原衬垫的这种作用在分析系统中使得剂量响应曲线更加陡峭，但也减少了灵敏度。这是由固定和自由的抗原对标记抗体的竞争结合导致的。对于一个浓度分布狭窄的确定的分析物，各种各样的剂量响应图形可能对未知样本的准确估计有必要。

Besides analysis, the concept of membrane immunochromatography can be expanded to in situ fractionation of chemical species. Transport of a specimen mixture through membrane pores may lead to the complete separation of analyte from other interfering substances provided a suitable antigen–antibody reaction system is employed. We have demonstrated isolation of single-class plasma lipoproteins based on the immunological method associated with streptavidin– biotin capture technology, and the lipoprotein cholesterol was consecutively quantified on the same strip without handling of reagents (11).

    除了分析，膜免疫层析的原理还能延伸到化学样本的原位分级分离。样本混合物通过膜孔的传输使得分析物从其他干扰物中完全分离，因此提供了合适的抗原抗体反应。我们还论证了基于免疫学方法结合链亲和素捕获技术分离单一级别血浆脂蛋白，并且在同一条带上不用对反应物进行操作就能连续定量检测脂蛋白胆固醇。

Antibody Coupling to Signal Generator(抗体在信号发生物上的结合)

Labels that do not require additional procedures (e.g., enzyme reactions) for signal generation are preferred in one-step chromatographic assay. Latex beads made of organic polymers comprising dyes of diverse colors have been conventionally used as signal generators in a rapid assay (3, 7, 12, 13). Bead size in a range from several nanometers to hundreds of micrometers are commercially available. Attachment of antibody to the bead surface can be accomplished by either physical adsorption or covalent binding depending on the exterior property and the presence of a functional group (mostly an amino or carboxyl group). Another label in an extensive spread is gold colloids that can be synthesized in the laboratory by reducing AuCl2 (14, 15). According to different reducing agents and their concentrations, the particle size produced varies between approximately 1 and 100 nm—the larger the size, the higher the signaling efficiency. For conjugation, immunoglobulin is directly adsorbed on the particle surfaces, mediated mainly by London–van der Waals force and hydrophobic interaction. Liposome is also another potential marker for the assay (16, 17). The lipid envelope containing dye molecules or other tracing substances releases them after disintegration by detergent and, thus, generates an amplified signal. Antibody can be linked to liposome particles by covalent binding.

   在一步层析检测中，不需要额外程序（如酶反应）来产生信号的标记是首选的。在快速检测中，有机聚合物制成的并包含有各种颜色染色的乳胶珠作为传统的信号产生物被使用过。尺寸范围从几纳米到几百微米的珠粒在市场上都有存在。抗体结合到珠粒表面可以通过物理吸附或共价结合（这要有表面特性和功能团（主要是氨基或羧基）的存在）来完成。另一个有广泛应用的是胶体金，在实验室能用还原AuCl₂来制取。根据不同的还原试剂和试剂浓度，产生的颗粒大小在大约1nm和100nm之间变化，尺寸越大，信号效果越高。对于结合，免疫球蛋白直接吸附在颗粒的表面上，主要通过London－范德华力和疏水相互作用。脂质体也是另外一个用于检测的有潜力的标记。脂质膜包含染色分子或其他追踪物质，被去污剂瓦解后释放它们，因而产生了一个强化的信号。抗体能通过共价键结合在脂质体颗粒上。

With colloidal gold as an example, labeling of detection antibody can be performed with high reproducibility under predetermined conditions. The colloids are formed in solution by virtue of a balance between electrostatic repulsion and London–van der Waals attraction among each particle (18, 19). However, on addition of ionic substances (e.g., NaCl), the attracting force becomes greater than the counteraction, which leads to an aggregation accompanying a color change from red (maximum absorbance at 520 nm, A520) to blue (at 580 nm, A580) (20). This instability can be prevented by coating the colloidal surfaces with protein molecules such as immunoglobulin. Optimal conditions of pH and antibody concentration for the coating can be determined by measuring the differential absorbance, i.e., A520–A580 (Fig. 2). For goat antibody, a peak A520–A580 was obtained in the pH range 9 to 10 (Fig. 2, upper panel). In general, a protein maximally adsorbs on the gold surface at the isoelectric point (pI) of the molecule or 0.5 pH unit higher (18, 19). In contrast, there is a minimal antibody concentration (approximately, 100 mg/mL in Fig. 2, lower panel) that can stabilize the gold particles.

     以胶体金为例，检测抗体的标记可以在预定的条件下高重复性进行。胶体金在溶液中是通过每个颗粒间静电排斥和London－范德华吸引力之间的平衡这个优点形成的。然而，当加入离子物质（如NaCl），吸引力比排斥力更大些，这使得颗粒聚集伴随着颜色从红色（最大吸附在520nm，A520）改变到蓝色（在580nm，A580）。这种不稳定性可以通过在胶体表面覆盖蛋白分子比如免疫球蛋白来抑制。检测差异吸光度，即A520－A580（Fig.2），可以确定覆盖反应的最佳PH值和抗体浓度。以羊抗体为例，在PH范围9－10得到A520－A580顶点（Fig.2,上图）。一般的说，蛋白在金表面最大的吸附在分子的等电点（pI）处或再高0.5个PH值处。相反地，存在一个最小抗体浓度（在Fig.2中大约100μg/ml，下图）能稳定金颗粒。

The performance of the tracer depends on external chemical properties of gold particles in addition to the affinity of the detection antibody. Since the residual gold surfaces, after binding to immunoglobulin, cause an interaction between the particle and solid matrix as well as the colloids themselves, they should be covered with an inert substance (21). Different substances create variable chemical environments (such as charge distribution on the outside) that may control the degree of nonspecific interactions of the gold colloids. For instance, casein as a blocking agent produces a dominant negative charge at neutral pH, whereas gelatin produces net positive ions. On the contrary, bovine serum albumin and polyethylene glycol produce conditions of neutrality without net charges. In our experience, casein in many immunoassay applications yielded the highest signal-to-noise ratio as a consequence of background suppression (22, 23) although distinct systems may demand separate optimal conditions.

    追踪物的性能除了依赖于检测抗体的亲和性外，还依赖于金颗粒的表面化学性能。因为结合免疫球蛋白之后还存在残留的金表面，会导致颗粒和固体介质或胶体本身之间产生相互作用，所以它们需要用惰性物质结合。不同物质产生不同的化学环境（例如外界的电荷分布），这可能会控制着胶体金非特异相互作用的程度。例如，酪蛋白作为封闭剂在中性PH产生明显的负离子，而白明胶产生净正离子，相反的，牛血清蛋白和聚乙二醇产生中性条件没有净电荷。以我们的经验，在很多免疫检测应用中酪蛋白通过抑制背景，产生最高的信号－噪声比例，尽管在不同的系统中可能要求各自最佳条件。

Preparation of Membrane with Immobilized Antibody(固定有抗体的膜的准备)

The capture of the labeled antibody–analyte complex for signal generation can be achieved with the antibody immobilized in a defined area of nitrocellulose membrane as mentioned. Two immobilization methods widely used are physical adsorption and chemical binding. The membrane is hydrophobic and, thus, lends itself for protein adsorption that is carried out by simply transferring the antibody solution onto the surface (9). The method, however, allows neither a firm immobilization of antibody on the solid surface during incubation procedure nor proper orientation of the antibody molecules for complex formation with antigen. Potential dissociation of the binder can be corrected by using covalent immobilization (24, 25). Crosslinking reagents such as glutaraldehyde (26, 27), the simplest form, may offer stable coupling of the immunoreactant to the membrane surface. This has been accomplished by physically coating the reagent in a monolayer, removing the excess chemical, immobilizing immunoglobulin, and inactivating the remaining functional groups. Adjustment of the positioning of the binder molecules is feasible by means of site-directed immobilization via chemical modification of the carbohydrate chain (24, 28, 29). Covalent binding of antibody is particularly beneficial if a detergent has to be used as the blocking agent of the residual surfaces. The chemical blocker is known to readily dissociate the antibody molecules physically adsorbed (30).

   对产生信号的标记抗体－抗原复合物的捕获，是用抗体固定在已经提到的硝酸纤维素的特定区域内来达到的。两种广泛用到的固定化方法是物理吸附和化学连接。膜是疏水的，因而可以用于蛋白吸附，只用简单的将抗体溶液放在表面就能实现。然而这种方法使得固相表面形成的固定化抗体在温育中并不牢固，在与抗原结合形成过程中也没有合适的抗体方位。结合物潜在的分裂可以用共价固定来解决。交联试剂如戊二醛，最简单的结构，可使免疫反应物在膜表面稳定联结。这可通过物理地铺上单层试剂，出去多余的化学试剂，固定化免疫球蛋白，然后失活残留的功能团这一过程来完成的。结合物分子位置可以用经碳水化合物链的化学修改的定点固定化方法来调节。如果去污剂作为残留表面的封闭剂，那么抗体的共价结合就特别有用。大家都知道，化学封闭剂能将物理吸附的抗体游离。 

Accumulated data in our laboratory showed that immobilization of antibody directly to different membranes (i.e., pore size, manufacturer, or fabrication process) produced a large variation in the protein binding capacity. This could give rise to problems in controlling qualities for quantitative analytical systems. However, these problems have been overcome successfully by introducing a monolayer of mediator (e.g., protein, homopolyamino acid) underneath the antibody coating. Various surface modifiers can be used for distinct patterns of signal generation (see Fig. 3) that are closely related to the detection mode desired. When glutaraldehyde was used as a crosslinker for chemical immobilization, the signal density was directly proportional to the number of functional amino groups available on the modifier molecule. For instance, polylysine gave a signal concentrated in a confined region of the signal pad (Fig. 3, left), whereas casein with fewer amino groups exposed to liquid medium resulted in a weak color dispersed over the entire surface (Fig. 3, right).

     我们实验室中积累起来的数据显示抗体在不同膜（即孔径、产家或制造工艺）上直接的固定化，导致在蛋白质结合性能方面产生大量的差异。这可能会在用于定量分析系统的控制量上引发问题。然而这些困难已经通过在抗体层下引进一单层介质（如蛋白、同聚氨酸）成功克服了。不同的表面修改剂能被用于不同类型的信号产生物（Fig.3），这跟想要的检测模式紧密相关。当戊二醛作为交联剂用于化学固定时，信号密度直接与修改剂上的功能氨基团数目成比例。例如，多溶素使信号衬垫的信号在一定区域内集中，而酪蛋白，更少氨基团暴露在液体介质中，导致分散在整个表面的色彩黯淡（Fig.3右）。 

The intensity of signal as a response to an analyte concentration can be controlled by adjusting the concentration  of antibody added. Since the antigen– antibody reaction is a dynamic, equilibrium process, the immune complex formation increases in proportion to the number of accessible binding sites on the surface (25, 31). As the amount of antibody introduced increases, the complex density soon reaches a maximum  and remains approximately constant thereafter, although the antibody molecules immobilized continue to increase (31). Immunoglobulin that does not participate in the reaction may only contribute to the development of the noise, i.e., the background color at zero dose of analyte, as a result of nonspecific interaction between the antibody molecules immobilized and those labeled (32). Because the signal-to-noise ratio is an essential indicator of system performance, there should be an optimal concentration of antibody for a given immunoassay system.

    信号亮度是对分析物浓度的一个反映，能通过调节加入抗体的浓度来控制。因为抗体－抗原反应是一个动态、平衡的过程，所以免疫复合物的形成与表面上能到达的结合位点数成比例增长。当引入的抗体数增加，复合浓度很快就达到最大，从此保持接近常数，尽管固定的抗体分子继续增加。不参加反应的免疫球蛋白可能只对噪声的增加有贡献，即在0剂量分析物时的背景颜色，这由固定的和标记的抗体分子之间的非特异性作用导致的。因为信号－噪声比例是系统性能的本质指示，所以对一个给定的免疫检测系统来说存在一个最佳的抗体浓度。

The noise produced because of the presence of the surplus antibody can be further regulated after fabricating the signal pad. The causes of the background signal are typically cross-reactions between the two antibodies, used for capture and detection, and the nonspecific binding as mentioned. The cross-reacting molecules are usually eliminated prior to use by purging them in an appropriate affinity chromatographic column (33). The nonspecific binding produced a molecular contact for interaction (22, 32) and, thus, a process inducing a structural modification of the immobilized binder may be effective for diminishing the level of discord. Treatment with thermal energy (baking) can selectively alter the structure while minimizing impairment of the paratopes of antibody (Fig. 4). The process time of membrane baking can vary according to the character of the substance and its concentration left within the membrane pores after desiccation. If the substance stabilizes the protein against heat, optimal baking time would be extended in proportion to the concentration (see the effect of arginine in Fig. 4). On the contrary, when the material is less protective of immunoglobulin from the heat energy, the handling time may become too short to control the effectiveness reproducibly (e.g., methylcellulose as shown in Fig. 4).

    因多余抗体存在而产生的噪声，可在制作信号衬垫后再进一步的校准。背景信号的产生原因有两个用于捕获和检测的抗体间典型的交叉反应，和提到过的非特异性结合。事先在一个适合的亲和色谱里做清洗处理，一般就能除掉交叉反应分子。非特异性结合产生导致相互作用的分子间接触，因此一个能诱导固定抗体结构变化的过程可能对减少噪声的水平有效。经过热处理（烘焙）能选择地改变结构，从而减少抗体中抗体决定基的损失（Fig.4）。膜烘焙过程的时间根据物质的性能和干燥后留在膜孔内的物质浓度变化。如果物质能使蛋白抗热，则最佳烘焙时间与浓度成比例（见Fig.4中精氨酸的影响）。相反地，当物质较少保护免疫球蛋白受热，操作时间可能变得太短而不能克重复地控制效率（如Fig.4中的甲基纤维素）。

Chemical Settings Guiding Antigen–Antibody Reaction(抗原－抗体反应的化学性设定指南)

The binding reaction between antigen and antibody can be enhanced by optimizing chemical environments, such as acidity and ionic strength, to which the immunoreactants are exposed (34, 35). These chemical factors may balance the charge state of protein molecules and, thus, increase the degree of interaction between the reactants. In the chromatographic assays, the factors adjusted can also accelerate the transfer rate of the migrating components through the membrane pores, the surface of which is usually coated with an inert protein (e.g., bovine serum albumin and casein) to minimize undesirable nonspecific binding. The process of transport brings about the formation of encounter complex, the step prior to the binding reaction with the immobilized immunoglobulin (36). We furthermore found that an appropriate buffer ion can intensify the binding although its concentration and acidity remain constant (8). 

    抗原和抗体间的结合反应可通过优化免疫反应物暴露的化学环境，例如酸度和离子强度，来提高优化。这些化学因素可能会平衡蛋白分子的电荷状态，从而增加了反应物间相互作用的程度。在免疫层析检测中，调整过的因素可能还会提高流动组分通过膜孔的流动速度，在膜孔的表面一般都铺有惰性蛋白（如牛血清蛋白和酪蛋白）用来减少不想要的非特异性结合。传递过程会引发偶然复合物的形成，这步在与固定免疫球蛋白结合反应之前进行。我们进一步发现适合的缓冲离子能强化结合，虽然它的浓度和酸度都保持不变。

To further direct the reaction, agents protecting the immunocomponents from nonspecific binding are added to the medium carrying analyte (22, 37). Typically, an inert protein alone or in the combination with a mild detergent such as Tween 20 is used. The agents may not only block adsorption sites on the solid surface, but also interact with the reactants to produce proper antigen–antibody binding at the liquid–solid interface. This implies that analytical systems consisting of different constituents could require various settings of inert protein for each assay. Inert proteins most often used for immunoassays are casein (or in the form of nonfat dry milk), bovine serum albumin, gelatin, and ovalbumin (37). In certain systems, nonprotein agents such as polyvinylpyrrolidone and polyvinyl alcohol have been used. 

    为进一步指导反应，用于保护免疫组分避免非特异性结合的试剂，加入到含有分析物的介质中。一般地用到单独的惰性蛋白或结合有温柔去污剂如Tween20的。这些试剂不仅能封闭固相表面上的吸附位点，还能和反应物相互作用，从而在固液界面产生适当的抗原－抗体结合物。这意味着由不同组分组成的分析系统需要对用于每个检测的惰性蛋白进行不同的设定。经常用于免疫检测的惰性蛋白大多都是酪蛋白（或无脂干奶粉形式）、牛血清蛋白、明胶和卵清蛋白。某些系统还用过非蛋白试剂如聚乙烯吡咯烷酮和聚乙烯醇。

Design of Immunochromatographic Assay System(免疫层析检测系统的设计) 

A highly sensitive chromatographic system is desirable particularly for monitoring analyte present in trace concentrations within samples. Because the analyte molecules are recognized by forming the binding complex with antibody, a decrease in the detection limit is achieved only by an increase in the reaction yield. With the immunoreagents given, the reaction in an assay can be increased by properly designing the analytical system, for instance, by rearranging the system constituents prepared, adjusting their relative amounts, introducing a novel functional pad(s), and selecting the pad materials and dimensions. The scale of these design factors is variable depending on the assay performance required for analyzing different compounds.

    高灵敏的层析系统是一个理想的系统，特别对监测存在于样本追踪浓度的分析物。因为分析物分子能通过与抗体形成结合物而被发现，所以降低检测极限只能通过增加反应产率来达到。给定免疫试剂后，检测中的反应可以通过合理的设计分析系统来提高，例如，通过重新布置准备的系统中的组分，调节他们相对量，引进一个新的功能性衬垫，选择衬垫的材料和尺寸等等。这些设计要素的比例依赖于分析不同化合物的检测效能。

To achieve high sensitivity, the minimum concentration of analyte detectable should be reproducibly measured by a signal distinguishable from the background, i.e., noise at zero dose of analyte (38). The signal-tonoise ratio, thus, may represent the assay sensitivity that can be modulated by either enhancement of signal or suppression of noise. Because the antigen–antibody reaction is an equilibrium binding reaction, the magnitude of the signal relies on the concentration of each reactant. The signal can be amplified by increasing either of the two antibodies for detection and capture, but it soon reaches a higher limit. Thereafter, the background signal also develops due to interactions between protein molecules as described. In determining the optimal concentration of each antibody, we have used a strategy in which the capture antibody is first immobilized at a particular density on the predetermined site of membrane, and the labeled antibody is then varied to retain a maximum signal-to-noise ratio. Deviation in the immobilized antibody density is relatively high even with a minute change in experimental conditions, and the amount of detection antibody can be accurately controlled by transferring a metered volume (8).

     为了达到高灵敏度，通过产生的信号与背景不同，即0剂量分析物的噪音，应该可重复的测到能检测到的最小分析物浓度。从而，信号－噪音比例能代表检测灵敏度，这样灵敏度就可通过强化信号或抑制噪音来调节。因为抗原－抗体反应是一个平衡结合反应，所以信号量依赖于每个反应物的浓度。通过增加用于检测和捕获的两个抗体的其中一个，信号就能被强化，但是很快就达到了更高的极限。此后背景信号也会因为此前提到过得蛋白间的相互作用，而得到提高。在测定每个抗体的最佳浓度中，我们用到了这样一个方案：先将捕获抗体以特定的浓度固定在膜上预留的部位，然后改变标记抗体浓度得到最大的信号－噪音比例。尽管在实验条件下只有很小改变，固定抗体的浓度偏差也会相对较高，而检测抗体的量就能通过移取量好的体积来精确控制。 

Because of the demand for a characteristically short assay time, immunochromatography is driven by nonequilibrium conditions (8), and the time assigned for the antigen–antibody reaction, as a consequence, becomes a controlling factor in forming the binding complex. To tune up system performance without a significant change in total assay time, we suggest two physical modifications: (1) variation of the thickness of glass pad on which the labeled antibody is deposited in a dry state and (2) installation of an additional reaction pad (see Fig. 1B). In an immunostrip (Fig. 1), the analyte-containing medium absorbed from the bottom end of the glass pad instantaneously dissolves the labeled antibody, and transfers the mixture into the nitrocellulose membrane connected in the upper position. Since the flow rate in the nitrocellulose membrane is much slower than that through the glass pad, the material transfer to the upper compartment is the rate-limiting step. This permits the formation of binding complex between the labeled antibody and analyte in the void space of glass fibers. Although a larger volume of the pad would result in a longer reaction time, it may be counteracted by a reactant dilution causing a decreased signal and also an unacceptably long assay (8). Alternatively, an additional spacer for extending the reaction time can be built in between the glass pad and the membrane for signal generation as described above. The pad length should be balanced toward both the signal intensity and the test time.

    由于短检测时间这个特征的需求，免疫层析要在非平衡条件下进行，使得用于抗原－抗体反应的时间成为形成结合物的控制因素。为了调高系统性能，而不在总检测时间上有大的改变，我们建议两个物理性修改：（1）改变放有干燥标记抗体的衬垫的厚度，（2）安装额外反应衬垫（见Fig.1B）。在一个免疫条中（Fig.1），含有分析物的溶液从底端的玻璃衬垫被吸附上来，即刻溶解了标记抗体，并使混合物移进连接在高位的硝酸纤维素膜里。因为在膜中的流速要比通过玻璃衬垫的要慢，所以溶液向上层流动是限速步骤。这就使得标记抗体和分析物间结合物的形成在玻璃纤维的空的空间里进行的。更大的衬垫体积会导致更长的反应时间，也会因反应物的稀释造成信号减弱，并且是个不能接受的长检测。此外，还可以在玻璃衬垫和用于产生信号的膜之间安装一个额外的垫片来延长反应时间。衬垫的长度应该在信号亮度和检测时间之间寻找平衡。

Thermal Stabilization(热稳定)

The shelf-life of fabricated immunostrips is determined primarily by the stability of antibody molecules which tend naturally to be inactivated by thermal energy. The inactivation rate depends directly on exposure temperature (37, 39), and it is important to investigate a method of protein stabilization and to estimate the shelf-life of the strip, i.e., the duration for maintaining 90% of the initial activity.

    制作完成的免疫条的货架寿命主要由抗体分子的稳定性来决定，抗体在受热情况下会自然失活。失活速度直接决定于暴露的温度，因而研究稳定蛋白的方法和测量免疫条的货架寿命，即保持最初活性90％的持续时间，是很重要的。

Because the inactivation of immunoglobulin is caused mainly by a change in the structural conformation during dehydration and storage, its prevention can be accomplished by handling the molecules in the presence of a stabilizer (37). In general, functional proteins with an added stabilizing agent, such as bovine serum albumin or casein, can be protected from various damaging factors (e.g., free radicals, proteolytic activity, heavy metals) remaining in the environment. Similar effects are also obtained by using animal serum (or human serum) and amino acids (40, 41). When protein is dehydrated, water molecules that were incorporated within the protein structure are dissociated, inducing structural instability in the protein (42–45). This may be overcome by replenishing the sites with sugar molecules such as trehalose and sucrose.

    因为免疫球蛋白的失活主要是由在脱水和贮存期间结构构造改变导致的，所以可以通过在稳定剂中对分子的处理来完成对它的抑制。一般来说，加入稳定剂如牛血清蛋白或酪蛋白，能使功能蛋白避免受到环境中的各种破坏性因子（如自由基，蛋白水解活性，重金属）的损害。类似的作用也可以用动物血清（或人血清）和氨基酸来达到。当蛋白脱水时，与蛋白结构结合在一起的水分子被分离，这会使蛋白结构不稳定。在位点上补充糖分子如海藻糖和蔗糖就能解决这个问题。

The immunostrips treated with various potential stabilizers produced significantly different signals in their intensities (Fig. 5). The order of substances showing higher stabilization was sugars, amino acids, and sera. In particular, a disaccharide, trehalose, furnished adequate protection of protein against heat under the conditions used. When a protein is desiccated in the presence of trehalose, water molecules bound to the protein structure by hydrogen bonding evaporate and the sites are then replaced by the sugar to maintain an intact confirmation (42–45). If the evaporation proceeds further, trehalose in solution turns to a form of syrup with high viscosity and then to a viscoelastic state. At this point, continual drying induces a phase change of the sugar to a glass state so that the protein molecules are surrounded by trehalose crystals, preserving them from thermal energy (46, 47). The stabilization effect was not significantly improved by using trehalose in combination with other substances (e.g., amino acids).

    经过各种有潜力的稳定剂处理的免疫条产生在亮度上有明显差异的信号（Fig.5）。表现出更高稳定的依次有糖类、氨基酸和血清。特别是二糖、海藻糖能提供蛋白足够的保护，对抗所在条件下的热量。当蛋白在海藻糖存在下干燥，经氢键结合在蛋白结构上的水分子蒸发掉，留下的位点随后被糖替代，确保了结构的完整。如果干燥得更深一步，海藻糖在溶液中就会形成高粘性得糖浆，随后并转变成粘弹性状态。这时候，连续干燥引起糖的相变，变成玻璃状态，这就使得蛋白分子被海藻糖包围，从而避免受热。海藻糖结合其他物质（如氨基酸）不会很大的改善稳定性能。 

Optimal concentrations of trehalose reconciled with the wicking rate of medium were 3% for the immobilized antibody and 24% for the gold–antibody conjugate lyophilized within a glass-fiber membrane pad. Under these conditions, the immunostrips were examined for shelf-life by performing thermal acceleration tests at a high temperature (Fig. 6). If the strips constructed with trehalose were exposed to 60°C, acceptable activity, i.e., higher than 90% of the initial activity, was retained for 21 days (Fig. 6, trehalose). This contrasted with the strips prepared in the absence of the sugar, which were active only less than 1 day (Tris). By comparing the result of stabilization with that reported (48), the shelf-life in the presence of trehalose is equivalent  to approximately 2 years at room temperature.

    中和介质的孔速率，海藻糖的最佳浓度是3％用于固定抗体，24％用于玻璃纤维膜垫中的干金抗体结合物。在这种情况下，通过在高温下热加速测试来测量免疫条的货架寿命（Fig.6）。如果将弄好的带有海藻糖的免疫条暴露在60℃下，可接受的活性，即高于初始活性的90％，能保持21天（Fig.6,海藻糖）。这跟没有糖的免疫条形成鲜明对比，它只在不到1天的时间里保持活性（Tris）。跟文献报道过的稳定性文论比较，有海藻糖的货架寿命相当于在室温下大约2年。

CONCLUDING REMARKS(结论) 　

An immunochromatographic assay system can be constructed in the laboratory by preparing two major components, the detection antibody labeled with a signal generator and the capture antibody immobilized on the surfaces of NC membrane strip, arranging them under optimal chemical and physical settings, and fi- nally preserving immunoglobulin in the presence of stabilizers. The detection capability of such constructed membrane strips can be varied by changing: (a) the concentrations of the two antibodies, (b) the agents for blocking residual membrane surfaces, (c) the chemical composition (e.g., buffer ions and protective substances against nonspecific binding) of the aqueous medium, (d) the dimensions of the tracer-releasing membrane, and (e) the introduction of a time-delay pad for the antigen–antibody reaction. Even after assembling the system, the performance can be corrected by structurally modifying the antibody molecules by applying thermal energy.

    免疫层析检测系统能在实验室里制造出来，先准备两个主要的组分，标有信号产生物的检测抗体和固定在NC膜条表面的捕获抗体，然后在最佳的化学和物理的条件下安置它们，最后在有稳定剂的存在下保存免疫球蛋白。这样构建的膜条的检测能力可以通过改变：（a）两个抗体的浓度，（b）用于封闭残留的膜表面的试剂，（c）水溶液中的化学成分（如缓冲离子和对抗非特异性结合的保护物质），（d）追踪物－释放的膜的尺寸和（e）引入用于抗原抗体反应的延时衬垫来改变。甚至在组装系统之后，它的性能还能通过应用热能量结构性修改抗体分子来校正。

The analytical method is applicable to many different fields of diagnosis requiring immediate, on-site examination. Its medical utilization includes a large group of target analytes (e.g., hormones, protein indicators, infectious factors) that can be measured either in a doctor’s office or at points of care such as an emergency room and the patient’s bedside. If determination of an analyte concentration is required, the assay system based on immunochromatography can be combined with a detector that is capable of precisely quantifying the binding complex formed as a result of the test, although the system is commonly fabricated in a stand-alone format. Environmental monitoring is another potentially large area for using immunoassays including detection of noxious substances such as pesticides, insecticides, and bacteria in drinking water. Likewise, food contaminants are also analytes demanding instantaneous measurement using the same assay protocol.

    这种分析方法适用于很多不同地点的需要即时就地的诊断检测。它的医学利用包括很多种目标分析物（如激素、蛋白指示剂、传染因子），它们可以在医生的办公室或医疗地点如急诊室和病人的床边。如果需要测量分析物的浓度，建立在免疫层析基础上的检测系统可与监测器相结合，这个监测器能准确定量形成的作为测试结果的结合物，虽然系统一般以单独站立的形式制造出来。环境监控也是另一个潜在的需要免疫检测的大领域，包括检测有害物质如杀虫剂和饮水中的细菌。同样食品污染物也是需要即时检测的，使用相同的检测方案的检测。